WO2019234221A1

WO2019234221A1 - Methods for stratification and treatment of a patient suffering from chronic lymphocytic leukemia

Info

Publication number: WO2019234221A1
Application number: PCT/EP2019/064946
Authority: WO
Inventors: Christine Le Roy; Nadine Varin-Blank; Arsène MEKINIAN; Marouane BOUBAYA; Vincent LEVY; Florence AJCHENBAUM-CYMBALISTA; Anne QUINQUENEL
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Université Paris Xiii Paris-Nord; Assistance Publique-Hôpitaux De Paris (Aphp)
Priority date: 2018-06-08
Filing date: 2019-06-07
Publication date: 2019-12-12

Abstract

The present invention relates to methods for stratification and treatment of progressive chronic lymphocytic in a patient in need thereof Depending on the cytokines produced, several regulatory B cell (Breg) subsets contribute to immune resolution and their enrichment leads to disease-associated immunosuppression. Chronic Lymphocytic Leukemia (CLL) is a heterogeneous clonal B cell neoplasm ranging from indolent to rapidly progressive clinical course that eludes tumor clearance. This study presents a comprehensive phenotypic and functional analysis of the regulatory subpopulations in CLL patients at various stages of the disease. Cytokines profiling of CLL-B cells evidences the production at various extents of IL-10 and TGFβ1. Remarkably, CLL cells express also the FOXP3 transcription factor, an original marker of regulatory T cells. The three proteins are produced by subpopulations with markers of activated and memory Β cells defining a specific signature. Based on CD5 and CD19 expression, intraclonal heterogeneity showed differential regulatory factors production relevant to disease evolution. Functional studies proved their regulatory capacities targeting T cell differentiation, proliferation and secretion. IL-10, TGFβ1 and FOXP3 expressions combined in a polyfunctional score strongly correlated with high-risk factors of progression. This profiling helps to predict progression and pinpoints immune dysfunction in CLL. Thus, the present invention relates to methods for diagnosing progressive chronic lymphocytic in a patient comprising a step of determining the level of IL-10, TGFβ1 and FOXP3 and methods for treating said patient.

Description

METHODS FOR STRATIFICATION AND TREATMENT OF A PATIENT SUFFERING FROM CHRONIC FYMPHOCYTIC FEUKEMIA

FIEFD OF THE INVENTION:

The present invention relates to methods for stratification and treatment of progressive chronic lymphocytic in a patient in need thereof

BACKGROUND OF THE INVENTION:

Chronic lymphocytic leukemia (CLL) is a lymphoid malignancy prevalent in the elderly presenting with a heterogeneous clinical course. Some patients experience a progressive disease with rapid poor outcome while others exhibit an indolent leukemia that does not impact life expectancy (1). The disease results in the clonal expansion of small, mature B lymphocytes, which accumulate in the bone marrow, blood and secondary lymphoid organs. In progressive patients chemo- and immunotherapies are not curative and residual clonal cancer cells re- populate both lymphoid organs and peripheral blood; the antitumor immune surveillance being ineffective (1, 2). The heterogeneity of CLL has been investigated and related to immunophenotypic markers including CD5, CD 19 or CD38 expressions. Moreover, genetic studies have pointed out the importance of stereotyped CDR3 sequences of immunoglobulin heavy chain variable region (IGHV) genes and of antigen receptor (BCR)-triggered pathways leading to apoptotic defect (3, 4). These membrane and signaling features converge to an active antigen-driven selection in the clonal expansion of the malignant cells. Indeed, functional studies have confirmed the relevance of signals propagated by BCR triggering in the heterogeneity of leukemic cell survival in vitro. BCR effectors such as Spleen tyrosine kinase, Phospholipase C g2 or NFAT transcription factors are often overexpressed and/or constitutively activated in B-CLL cells and allow the expression of target genes important for cell survival (5, 6). Expression of unconventional activators, such as the zeta chain associated protein (ZAP-70) commonly expressed in T and NK cells, is also associated with increased cell survival. In addition, the capacity to generate and propagate such activating signals at the cellular level correlates with disease progression. By contrast, defective signal transduction is prevalent in indolent disease and related to an anergic phenotype of the leukemic cells (7, 8). The important driving force of BCR initiated signals is especially encountered in lymph nodes, which are important sites for antigen recognition and account for the accumulation of malignant cells in synergy with microenvironmental factors (9, 10). Besides heterogeneous signaling capacity, the malignancy is also characterized by an imbalance of the immune subpopulations present in peripheral blood and lymphoid organs of CLL patients. T cell subsets that account for immune surveillance in tumor development encompassing CD4⁺ helpers (Th) and regulatory T cells (Tregs) have an altered ratio in these hematological malignancies (1 1 , 12). Thus, Tregs are increased in CLL and correlate with several clinical/biological features of progressive disease, whereas CD8⁺ T cells from CLL patients show functional defect in proliferation and cytotoxicity, but preserved cytokine production reflecting T-cell exhaustion (13). Such impairment of immunological homeostasis is often observed when ineffective antitumor immunity takes place during neoplasm progression and has been attributed to the production of several regulatory molecules and cytokines by specific B cell subsets ascribed to regulatory B cells (14-17). Recent data have shown that these B cell subsets play an important role in the direct or indirect suppression of inflammatory response and in the maintenance of tolerance through the production of IL10 (18- 21). Various teams have undertaken the phenotypic characterization of these subsets both in murine models and in human pathologies. However, this strategy did not identify a unique consensus but several phenotypes of progenitor populations with suppressive activity (16). Among others, murine CD5⁺ Bla and CDld^hlghCD5⁺CDl9^hlgh B10 cells are IL-10 producing cells (22, 23). Studies unraveling the functional properties of the CD5⁺ Bla lineage have uncovered regulatory properties leading to bias of the immune cells repertoire amongst which expansion of the Treg population and suppression of Thl and Thl7 differentiation (24). Human Breg subsets, identified by their capacity to suppress Thl differentiation and convert CD4⁺ T cells into Tregs via IL10 production, have also been described (25, 26). In peripheral blood from both healthy individuals and patients with autoimmune diseases or neoplasms, various IL10 producing subtypes have been reported (26). The latters include CDl9⁺CD24^hlghCD38^hlgh immature B cells, CD 19⁺CD24^highCD27⁺ B10 cells,

CD 19⁺CD38⁺CD 1 d⁺IgM⁺CD 147⁺Granzyme⁺ B cells and CD27^intermediateCD38^high plasmablasts (19, 27, 28). Induction of IL10 in the various subsets seems to require signals from activated CD4⁺T cells with CD40L playing a major stimulatory role, while IL21 -dependant signals rather induce Granzyme (GrB) producing regulatory B cells preferentially (29). Additional induced Bregs can also exert a suppressive mechanism via the production of TGF 1 and indoleamine- 2,3 dioxygenase (IDO) (30).

Remarkably, the majority of these phenotypic features are commonly observed in B- CLL populations irrespective of their IGHV mutational status. Due to the expression of CD5, CLL-B cells have been hypothesized for long as being derived from a human Bl lineage recognizing natural antibodies (31, 32). At present, CLL-B cells are considered as antigen- experienced B cells with an IGHV mutational status reflecting a T-dependent (mutated IGHV, M-IGHV) or T-independent (unmutated IGHV, UM-IGHV) memory phenotype with expression of CD27 in both cases (1). CLL malignant B cells have a clear survival advantage over the other normal B cells (33). Furthermore, diverse triggering events have been shown to induce IL10 in these cells, which share immunosuppressive capacities with B10 cells (21).

In the present invention, the inventors systematically analyzed the capacity for CLL cells from donors with indolent and progressive disease to produce in response to BCR/CD40 triggering key cytokines involved in immune modulation and in tumor surveillance. We found that a subset of leukemic cells of variable frequency produced constitutive IL10 and induced TGF 1 . Those cells also expressed the transcription factor FOXP3 that is a hallmark of Tregs. Cells that produce these three regulatory proteins display a specific signature somehow different from the already described B10 cells. Evidently, these cells share the regulatory properties attributed to Bregs toward CD4⁺ T cells. Finally, we give evidences that the combination of the three factors in a polyfunctional score is indicative of progression and might be used for the stratification of the disease. These novel findings provide also important insights on how CLL cells modify their immunological environment to their advantage.

SUMMARY OF THE INVENTION:

The present invention relates to methods for stratification and treatment of progressive chronic lymphocytic in a patient in need thereof.

In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

Depending on the cytokines produced, several regulatory B cell (Breg) subsets contribute to immune resolution and their enrichment leads to disease-associated immunosuppression. Chronic Lymphocytic Leukemia (CLL) is a heterogeneous clonal B cell neoplasm ranging from indolent to rapidly progressive clinical course that eludes tumor clearance. This study presents a comprehensive phenotypic and functional analysis of the regulatory subpopulations in CLL patients at various stages of the disease. Cytokines profiling of CLL-B cells evidences the production at various extents of IL-10 and TGFp 1 . Remarkably, CLL cells express also the FOXP3 transcription factor, an original marker of regulatory T cells. The three proteins are produced by subpopulations with markers of activated and memory B cells defining a specific signature. Based on CD5 and CD 19 expression, intraclonal heterogeneity showed differential regulatory factors production relevant to disease evolution. Functional studies proved their regulatory capacities targeting T cell differentiation, proliferation and secretion. IL-10, TGFp i and FOXP3 expressions combined in a polyfunctional score strongly correlated with high-risk factors of progression. This profiling helps to predict progression and pinpoints immune dysfunction in CLL.

Accordingly, the invention relates to a method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment comprising a step of determining the level of IL-10, TGFp i and FOXP3 in a biological sample obtained from the patient.

The method of the invention may further comprises a step consisting of comparing the level of IL-10, TGFp i and FOXP3 in the biological sample with a reference value, wherein detecting differential in the level of IL-10, TGFp i and FOXP3 between the biological sample and the reference value is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia.

In some embodiments, the present invention relates to a method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of: i) determining the level of IL-10, TGFp i and FOXP3 in a biological sample obtained from the patient, ii) comparing the level determined at step i) with a reference value, wherein detecting differential in the level of IL-10, TGFp i and FOXP3 between the biological sample and the reference value is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia.

In some embodiments, the present invention relates to a method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of:

i) determining the level of IL-10, TGFp i and FOXP3 in a biological sample obtained from the patient at time tl,

ii) determining the level of IL-10, TGFp i and FOXP3 in a biological sample obtained from the patient at time t2, and

iii) an increase of the levels determined at step 1) and ii) between tl and t2 is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

i) determining the level of IL-10, TGFp 1 and FOXP3 in a biological sample obtained from the patient at time tl, ii) determining the level of IL-10, TGFp i and FOXP3 in a biological sample obtained from the patient at time t2,

iii) building a polyfunctional score combining the levels determined at step i) and a polyfunctional score combining the levels determined at step ii), and

iv) an increase of the polyfunctional score between tl and t2 is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

In further aspect, the method of the invention comprises a step consisting of determining the level of IL-10, TGFp i and FOXP3 in the biological sample, followed by an unsupervised hierarchical cluster analysis and an index called polyfunctional score that was built by combining IL10, TGFp i and FOXP3 expressions. An increase of the polyfunctional score between two points of the clinical follow up is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

In some embodiments, the present invention relates to a method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment comprising the steps of: i) determining the expression level of IL-10, TGFp 1 and FOXP3 in a biological sample obtained from the patient, ii) applying an unsupervised hierarchical cluster analysis iii) building a polyfunctional score combining IL-10, TGFp i and FOXP3 expression levels determined at step i), an increase of the polyfunctional score value between two points of the clinical follow up is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

As used herein, the term“patient” denotes a mammal. Typically, a patient according to the invention refers to any patient (preferably human) afflicted or at risk to be afflicted with chronic lymphocytic leukemia. The term“patient” also refers to any patient (preferably human) afflicted or at risk to be afflicted with progressive chronic lymphocytic leukemia

As used herein, the term "chronic lymphocytic leukemia” or“CLL” has its general meaning in the art and refers to Acute myeloid leukemia such as revised in the World Health Organisation Classification C91.1. The term“Chronic lymphocytic leukemia” also refers to a heterogeneous clonal B cell neoplasm ranging from indolent to rapidly progressive clinical course that eludes tumor clearance. The term“Chronic lymphocytic leukemia” also refers to a lymphoid malignancy prevalent in the elderly presenting with a heterogeneous clinical course. Some patients experience a progressive disease with rapid poor outcome while others exhibit an indolent leukemia that does not impact life expectancy (1). The disease results in the clonal expansion of small, mature B lymphocytes, which accumulate in the bone marrow, blood and secondary lymphoid organs.

As used herein the term“IL-10” has its general meaning in the art and refers to Interleukin 10.

As used herein the term“TGFp 1” has its general meaning in the art and refers to Transforming Growth Factor Beta 1.

As used herein the term“FOXP3” has its general meaning in the art and refers to Forkhead Box P3.

As used herein, the term“biological sample” refers to B cells, chronic lymphocytic leukemia B cells, bone marrow sample, lymphoid organ sample, blood sample, serum sample and plasma sample.

In a further aspect, the present invention relates to a method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of determining the level of cells expressing IL-10, cells expressing TGFp i , and cells expressing FOXP3 in a biological sample obtained from the patient.

The method of the invention may further comprises a step consisting of comparing the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 in the biological sample with a reference value, wherein detecting differential in the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 between the biological sample and the reference value is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia.

In some embodiment, the present invention relates to a method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of:

i) determining the level of cells expressing IL-10, cells expressing TGFp i , and cells expressing FOXP3 in a biological sample obtained from the patient,

ii) comparing the level determined at step i) with their corresponding predetermined reference value, and

iii) detecting differential between the level determined at step i) with the predetermined reference value is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia.

In some embodiments, the present invention relates to a method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of: i) determining the level of cells expressing IL-10, cells expressing TGFp i , and cells expressing FOXP3 in a biological sample obtained from the patient at time tl,

ii) determining the level of cells expressing IL-10, cells expressing TGFp i , and cells expressing FOXP3 in a biological sample obtained from the patient at time t2, and

i) determining the level of cells expressing IL-10, cells expressing TGFp i , and cells expressing FOXP3 in a biological sample obtained from the patient at time tl,

ii) determining the level of cells expressing IL-10, cells expressing TGFp i , and cells expressing FOXP3 in a biological sample obtained from the patient at time t2,

In a further aspect, the method of the invention may further comprises a step consisting of combining the proportion of cells expressing IL-10, cells expressing TGFp i , and cells expressing FOXP3 in the biological sample to build a polyfunctional score. An increase of the polyfunctional score value between two points of the clinical follow up is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

i) determining the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 in a biological sample obtained from the patient,

ii) applying an unsupervised hierarchical cluster analysis,

iii) building a polyfunctional score combining the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 determined at step i). An increase of the polyfunctional score between two points of the clinical follow up is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

In some embodiments, the cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 relates to cells expressing high level of IL-10, cells expressing high level of TGFp 1 , and cells expressing high level of FOXP3.

As used herein, the term“level of cells expressing a biomarker such as IL-10, TGFp 1 and FOXP3” has its general meaning in the art and refers to the quantity of cells expressing the biomarker or the number of cells expressing the biomarker. The term“level of cells expressing a biomarker” also refers to the density of cells expressing a biomarker. The term“level of cells expressing a biomarker” also refers to the percentage of cells expressing a biomarker. The term “level of cells expressing a biomarker” also refers to the level or percentage of B cells expressing a biomarker. The term“level of cells expressing a biomarker” also refers to the level or percentage of CLL B cells expressing a biomarker.

In some embodiments, the term“level of B cells expressing a biomarker” refers to the level or percentage of B cells expressing a biomarker compared to total B cells.

In some embodiments, the term“level of CLL B cells expressing a biomarker” refers to the level or percentage of CLL B cells expressing a bio marker compared to total CLL B cells.

As used herein, the“polyfunctional score” refers to a combined value of expression and the term“reference value” refers to a threshold value or a cut-off value. The setting of a single “polyfunctional score” or“reference value” thus allows discrimination between a progressive chronic lymphocytic leukemia patient and indolent chronic lymphocytic leukemia patient; discrimination between a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatments and a patient not having or not at risk of having or developing progressive chronic lymphocytic leukemia or not resistant to treatments; a poor and a good prognosis with respect to the aggressiveness, invasiveness and/or recurrence of chronic lymphocytic leukemia, cancer relapse and/or overall survival (OS) for a patient. Typically, a "threshold value", "cut-off value" or“polyfunctional score” can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Particularly, the person skilled in the art may compare the quantity (obtained according to the method of the invention) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the quantity (or ratio, or score) determined in a biological sample derived from one or more patients having chronic lymphocytic leukemia. Furthermore, retrospective measurement of the quantity (or ratio, or scores) in properly banked historical patient samples may be used in establishing these threshold values.

Predetermined reference values used for comparison may comprise “cut-off’ or “threshold” values that may be determined as described herein. Each reference (“cut-off’) or polyfunctional score value may be predetermined by carrying out a method comprising the steps of

a) providing a collection of samples (such as B cells, blood) from patients suffering of chronic lymphocytic leukemia;

b) determining the level of the bio markers or the level of cells expressing the bio markers for each sample contained in the collection provided at step a);

c) ranking the tumour samples according to said level;

d) classifying said samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their level determined,

e) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding chronic lymphocytic leukemia patient (i.e. indolent chronic lymphocytic leukemia or progressive chronic lymphocytic leukemia; the duration of the event-free survival (EFS), metastasis- free survival (MFS) or the overall survival (OS) or both);

f) for each pair of subsets of samples, obtaining a Kaplan Meier percentage of survival curve;

g) for each pair of subsets of samples calculating the statistical significance (p value) between both subsets;

h) selecting as reference value for the quantity of cells, the value of the level the biomarkers or the level of cells for which the p value is the smallest.

For example, the level of the biomarkers or the level of cells expressing the biomarkers has been assessed for 100 cancer samples of 100 patients. The 100 samples are ranked according to their level of the biomarkers or their level of cells expressing the biomarkers. Sample 1 has the best level of the biomarkers or level of cells expressing the biomarkers and sample 100 has the worst level of the biomarkers or level of cells expressing the biomarkers. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding chronic lymphocytic leukemia patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.

The reference value or polyfunctional score is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the level of the biomarkers or level of cells expressing the biomarkers corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of levels of the m biomarkers or levels of cells expressing the biomarkers.

In routine work, the reference value (cut-off value) or polyfunctional score may be used in the present method to discriminate progressive chronic lymphocytic leukemia samples and therefore the corresponding patients.

Kaplan-Meier curves of percentage of survival as a function of time are commonly to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.

The man skilled in the art also understands that the same technique of assessment of the level of the bio markers or level of cells expressing the bio markers should of course be used for obtaining the reference value or polyfunctional score and thereafter for assessment of the level of the biomarkers or level of cells expressing the biomarkers of a patient subjected to the method of the invention.

In another embodiment, a score which is a composite of the level of the biomarkers or level of cells expressing the biomarkers may also be determined and compared to a reference value wherein a difference between said score and said reference value is indicative of a patient having or at risk of having or developing progressive chronic lymphocytic leukemia.

In a particular embodiment, the score may be generated by a computer program.

In one embodiment, the reference value or the polyfunctional score may correspond to the level of the biomarkers or level of cells expressing the biomarkers determined in a sample associated with a patient having indolent chronic lymphocytic leukemia or a patient not having or not at risk of having or developing progressive chronic lymphocytic leukemia. Accordingly, a higher level of the biomarkers or level of cells expressing the biomarkers than the reference value or a highest level of the bio markers or level of cells expressing the bio markers in the unsupervised hierarchical cluster analysis is indicative of a patient having or at risk of having or developing progressive chronic lymphocytic leukemia, or resisting to treatments and a lower or equal level of the biomarkers or level of cells expressing the biomarkers than the reference value is indicative of a patient having indolent chronic lymphocytic leukemia or a patient not having or not at risk of having or developing progressive chronic lymphocytic leukemia.

In another embodiment, the reference value or the polyfunctional score may correspond to the level of the bio markers or level of cells expressing the bio markers determined in a sample associated with a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment. Accordingly, a higher or equal level of the biomarkers or level of cells expressing the biomarkers than the reference value or a highest level of the bio markers or level of cells expressing the bio markers in the unsupervised hierarchical cluster analysis is indicative of a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment, and a lower level of the bio markers or level of cells expressing the bio markers than the reference value is indicative of a patient having indolent chronic lymphocytic leukemia or a patient not having or not at risk of having or developing progressive chronic lymphocytic leukemia.

In a further aspect, the method of the invention is performed in at least two times tl and t2. In some embodiments, the method of the invention is performed at time tl and at time t2 different from time tl .

Accordingly, an increase of the level of the biomarkers, level of cells expressing the biomarkers or the polyfunctional score is indicative of a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment, and a stable or a decrease of the level of the biomarkers, level of cells expressing the biomarkers or the polyfunctional score is indicative of a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

In some embodiments, the method of the invention further comprises determining the expression level of at least one biomarker selected from the group consisting of pro- inflammatory cytokines and/or regulatory cytokines.

In some embodiments, the method of the invention further comprises determining the level of cells (B cells or CLL B cells) expressing at least one bio marker selected from the group consisting of pro -inflammatory cytokines and/or regulatory cytokines.

The term“pro -inflammatory cytokines” has its general meaning in the art and refers to cytokines or soluble factors that are excreted from immune cells, such as specific helper T cells, and that promote inflammation through pro-inflammatory signals such as IL 1 b, IL2, IL4 or IFNy.

The term“regulatory cytokines” has its general meaning in the art and refers to cytokines or soluble factors that secreted by cells involved in the remodeling of the immune response in normal or pathological environments such as IL6, IL8 or TNFa.

Accordingly, the equilibrium between positive and negative regulatory cytokines or level of cells expressing regulatory cytokines balancing toward a lower level of pro- inflammatory cytokines or level of cells expressing pro -inflammatory cytokines in favour of the negative regulatory cytokines is indicative of a patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

In some embodiments, a higher level of regulatory cytokines or level of cells expressing regulatory cytokines than the reference value and/or a lower level of pro -inflammatory cytokines or level of cells expressing pro -inflammatory cytokines than the reference value is indicative of a patient having or at risk of having or developing progressive chronic lymphocytic leukemia; and a lower level of regulatory cytokines or level of cells expressing regulatory cytokines than the reference value and/or a higher level of pro -inflammatory cytokines or level of cells expressing pro -inflammatory cytokines than the reference value is indicative of a patient having indolent chronic lymphocytic leukemia or a patient not having or not at risk of having or developing progressive chronic lymphocytic leukemia.

The level of cells expressing the bio markers is determined by any well-known method in the art. In some embodiments, the level of cells expressing the biomarkers is determined such as described in the example. In some embodiments, the level of cells expressing the biomarkers is determined by flow cytometry. In some embodiments, the level of cells expressing the biomarkers is determined by IHC or immunofluorescence.

For example, the quantification of the cells expressing the biomarkers is performed by contacting the biological sample with a binding partner (e.g. an antibody) specific for a cell biomarkers of said cells. Typically, the quantification of the cells expressing the biomarkers is performed by contacting the tissue tumour tissue sample with a binding partner (e.g. an antibody) specific for IL-10, TGFp 1 or FOXP3.

Typically, the level of cells expressing the bio markers is expressed as the percentage of the specific cells per total cells (set at 100%). In some embodiments, the level of cells expressing the biomarkers may also consist of the number of these cells that are counted per one unit of surface area, e.g. as the number of cells that are counted per mm² of surface area of tumour tissue sample. In some embodiments, the quantification of the cells expressing the biomarkers is performed by flow cytometry or Fluorescence-activated cell sorting (FACS). In some embodiments, the quantification of the cells expressing the bio markers is performed by flow cytometry such as described in the example.

The level of the biomarkers may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed nucleic acid or translated protein.

In one embodiment, the biomarker expression level is assessed by analyzing the expression of the protein translated from said gene. Said analysis can be assessed using an antibody (e.g., a radio-labeled, chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker.

Methods for measuring the expression level of a biomarker in a sample may be assessed by any of a wide variety of well-known methods from one of skill in the art for detecting expression of a protein including, but not limited to, direct methods like mass spectrometry- based quantification methods, protein microarray methods, enzyme immunoassay (EIA), radioimmunoassay (RIA), Immunohistochemistry (IHC), Western blot analysis, ELISA, Luminex, ELISPOT and enzyme linked immunoabsorbant assay and undirect methods based on detecting expression of corresponding messenger ribonucleic acids (mRNAs). The mRNA expression profile may be determined by any technology known by a man skilled in the art. In particular, each mRNA expression level may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative Polymerase Chain Reaction (qPCR), next generation sequencing and hybridization with a labelled probe.

Said direct analysis can be assessed by contacting the sample with a binding partner capable of selectively interacting with the biomarker present in the sample. The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal (e.g., a isotope-label, element-label, radio-labeled, chromophore- labeled, fluorophore-labeled, or enzyme-labeled antibody), an antibody derivative (e.g., an antibody conjugate with a substrate or with the protein or ligand of a protein of a protein/ligand pair (e.g., biotin-streptavidin)), or an antibody fragment (e.g., a single-chain antibody, an isolated antibody hypervariable domain, etc.) which binds specifically to the protein translated from the gene encoding for the biomarker of the invention. In another embodiment, the binding partner may be an aptamer. The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as an isotope, an element, a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as an isotope, an element, a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be produced with a specific isotope or a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited to radioactive atom for scintigraphic studies such as 1123, 1124, Inl l l, Rel86, Rel88, specific isotopes include but are not limited to 13C, 15N, 1261, 79Br, 8lBr.

The afore mentioned assays generally involve the binding of the binding partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidene fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, silicon wafers.

In a particular embodiment, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize said biomarker. A sample containing or suspected of containing said bio marker is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art such as Singulex, Quanterix, MSD, Bioscale, Cytof.

In one embodiment, an Enzyme-linked immunospot (ELISpot) method may be used. Typically, the sample is transferred to a plate which has been coated with the desired anti biomarker capture antibodies. Revelation is carried out with biotinylated secondary Abs and standard colorimetric or fluorimetric detection methods such as streptavidin-alkaline phosphatase and NBT-BCIP and the spots counted. In one embodiment, when multi-biomarker expression measurement is required, use of beads bearing binding partners of interest may be preferred. In a particular embodiment, the bead may be a cytometric bead for use in flow cytometry. Such beads may for example correspond to BD™ Cytometric Beads commercialized by BD Biosciences (San Jose, California). Typically, cytometric beads may be suitable for preparing a multiplexed bead assay. A multiplexed bead assay, such as, for example, the BD(TM) Cytometric Bead Array, is a series of spectrally discrete beads that can be used to capture and quantify soluble antigens. Typically, beads are labelled with one or more spectrally distinct fluorescent dyes, and detection is carried out using a multiplicity of photodetectors, one for each distinct dye to be detected. A number of methods of making and using sets of distinguishable beads have been described in the literature. These include beads distinguishable by size, wherein each size bead is coated with a different target-specific antibody (see e.g. Fulwyler and McHugh, 1990, Methods in Cell Biology 33:613-629), beads with two or more fluorescent dyes at varying concentrations, wherein the beads are identified by the levels of fluorescence dyes (see e.g. European Patent No. 0 126,450), and beads distinguishably labelled with two different dyes, wherein the beads are identified by separately measuring the fluorescence intensity of each of the dyes (see e.g. U.S. patent Nos. 4,499,052 and 4,717,655). Both one-dimensional and two-dimensional arrays for the simultaneous analysis of multiple antigens by flow cytometry are available commercially. Examples of one-dimensional arrays of singly dyed beads distinguishable by the level of fluorescence intensity include the BD(TM) Cytometric Bead Array (CBA) (BD Biosciences, San Jose, Calif.) and Cyto-Plex(TM) Flow Cytometry microspheres (Duke Scientific, Palo Alto, Calif.). An example of a two-dimensional array of beads distinguishable by a combination of fluorescence intensity (five levels) and size (two sizes) is the QuantumPlex(TM) microspheres (Bangs Laboratories, Fisher, Ind.). An example of a two- dimensional array of doubly-dyed beads distinguishable by the levels of fluorescence of each of the two dyes is described in Fulton et al. (1997, Clinical Chemistry 43(9): 1749-1756). The beads may be labelled with any fluorescent compound known in the art such as e.g. FITC (FL1), PE (FL2), fluorophores for use in the blue laser (e.g. PerCP, PE-Cy7, PE-Cy5, FL3 and APC or Cy5, FL4), fluorophores for use in the red, violet or UV laser (e.g. Pacific blue, pacific orange). In another particular embodiment, bead is a magnetic bead for use in magnetic separation. Magnetic beads are known to those of skill in the art. Typically, the magnetic bead is preferably made of a magnetic material selected from the group consisting of metals (e.g. ferrum, cobalt and nickel), an alloy thereof and an oxide thereof. In another particular embodiment, bead is bead that is dyed and magnetized. In one embodiment, protein microarray methods may be used. Typically, at least one antibody or aptamer directed against the biomarker is immobilized or grafted to an array(s), a solid or semi- so lid surface(s). A sample containing or suspected of containing the biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample. After a period of incubation of said sample with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried. After all, quantifying said biomarker may be achieved using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.

In another embodiment, the antibody or aptamer grafted on the array is labelled.

In another embodiment, reverse phase arrays may be used. Typically, at least one sample is immobilized or grafted to an array(s), a solid or semi- so lid surface(s). An antibody or aptamer against the suspected biomarker is then labelled with at least one isotope or one element or one fluorophore or one colorimetric tag that are not naturally contained in the tested sample. After a period of incubation of said antibody or aptamer with the array sufficient to allow the formation of antibody-antigen complexes, the array is then washed and dried. After all, detecting quantifying and counting by D-SIMS said biomarker containing said isotope or group of isotopes, and a reference natural element, and then calculating the isotopic ratio between the biomarker and the reference natural element may be achieved using any appropriate microarray scanner like fluorescence scanner, colorimetric scanner, SIMS (secondary ions mass spectrometry) scanner, maldi scanner, electromagnetic scanner or any technique allowing to quantify said labels.

In one embodiment, said direct analysis can also be assessed by mass Spectrometry. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches (DeSouza and Siu, 2012). Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labelingor proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, LTQ Orbitrap Velos, LTQ-MS/MS, a quantification based on extracted ion chromatogram EIC (progenesis LC-MS, Liquid chromatography-mass spectrometry) and then profile alignment to determine differential expression of the biomarker.

In another embodiment, the biomarker expression level is assessed by analyzing the expression of mRNA transcript or mRNA precursors, such as nascent RNA, of biomarker gene. Said analysis can be assessed by preparing mRNA/cDNA from cells in a sample from a subject, and hybridizing the mRNA/cDNA with a reference polynucleotide. The prepared mRNA/cDNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses, such as quantitative PCR (TaqMan), and probes arrays such as GeneChip(TM) DNA Arrays ( AFF YMETRIX) .

Advantageously, the analysis of the expression level of mRNA transcribed from the gene encoding for biomarkers involves the process of nucleic acid amplification, e. g., by RT- PCR (the experimental embodiment set forth in U. S. Patent No. 4,683, 202), ligase chain reaction (Barany, 1991), self- sustained sequence replication (Guatelli et al., 1990), transcriptional amplification system (Kwoh et al., 1989), Q-Beta Replicase (Lizardi et al., 1988), rolling circle replication (U. S. Patent No. 5,854, 033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

In a further aspect, the method of the present invention is suitable for monitoring chronic lymphocytic leukemia progression in a patient in need thereof.

In a further aspect, the method of the present invention is suitable for determining whether a patient is eligible or not to an anti-cancer treatment or an anti-cancer therapy. For example, when it is determined that the patient is having or at risk of having or developing progressive chronic lymphocytic leukemia then the physician can take the choice to administer the patient with an anti-cancer treatment. Typically, the treatment includes chemotherapy, radiotherapy, radio immunotherapy, immunotherapy and drugs suitable for the treatment and prevention of progressive chronic lymphocytic leukemia.

The term“anti-cancer treatment” or“anti-cancer therapy” has its general meaning in the art and refers to anti-cancer compounds used in anti-cancer therapy such as tyrosine kinase inhibitors, tyrosine kinase receptor (TKR) inhibitors, EGFR tyrosine kinase inhibitors, anti- EGFR compounds, anti-HER2 compounds, Vascular Endothelial Growth Factor Receptors (VEGFRs) pathway inhibitors, interferon therapy, alkylating agents, anti-metabolites, immunotherapeutic agents such as sipuleucel-T, Androgen deprivation therapy (ADT), Interferons (IFNs), Interleukins, radiotherapeutic agents (such as Ra223, Pb2l2) and chemotherapeutic agents such as described below.

The term“tyrosine kinase inhibitor” or“TKΊ” has its general meaning in the art and refers to any of a variety of therapeutic agents or drugs such as compounds inhibiting tyrosine kinase, tyrosine kinase receptor inhibitors (TKRI), EGFR tyrosine kinase inhibitors, EGFR antagonists. The term“tyrosine kinase inhibitor” or“TKI” has its general meaning in the art and refers to any of a variety of therapeutic agents or drugs that act as selective or non-selective inhibitors of receptor and/or non-receptor tyrosine kinases. Tyrosine kinase inhibitors and related compounds are well known in the art and described in U.S Patent Publication 2007/0254295, which is incorporated by reference herein in its entirety. It will be appreciated by one of skill in the art that a compound related to a tyrosine kinase inhibitor will recapitulate the effect of the tyrosine kinase inhibitor, e.g., the related compound will act on a different member of the tyrosine kinase signaling pathway to produce the same effect as would a tyrosine kinase inhibitor of that tyrosine kinase. Examples of tyrosine kinase inhibitors and related compounds suitable for use in methods of embodiments of the present invention include, but are not limited to Erlotinib, sunitinib (Sutent; SU11248), dasatinib (BMS-354825), PP2, BEZ235, saracatinib, gefitinib (Iressa), erlotinib (Tarceva; OSI-1774), lapatinib (GW572016; GW2016), canertinib (Cl 1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec; STI571), leflunomide (SU101), vandetanib (Zactima; ZD6474), MK-2206 (8-[4-aminocyclobutyl)phenyl]-9-phenyl-l,2,4-triazolo[3,4- f][l,6]naphthyridin-3(2H)-one hydrochloride) derivatives thereof, analogs thereof, and combinations thereof. Additional tyrosine kinase inhibitors and related compounds suitable for use in the present invention are described in, for example, U.S Patent Publication 2007/0254295, U.S. Pat. Nos. 5,618,829, 5,639,757, 5,728,868, 5,804,396, 6,100,254, 6,127,374, 6,245,759, 6,306,874, 6,313,138, 6,316,444, 6,329,380, 6,344,459, 6,420,382, 6,479,512, 6,498,165, 6,544,988, 6,562,818, 6,586,423, 6,586,424, 6,740,665, 6,794,393,

6,875,767, 6,927,293, and 6,958,340, all of which are incorporated by reference herein in their entirety. In certain embodiments, the tyrosine kinase inhibitor is a small molecule kinase inhibitor that has been orally administered and that has been the subject of at least one Phase I clinical trial, more particularly at least one Phase II clinical, even more particularly at least one Phase III clinical trial, and most particularly approved by the FDA for at least one hematological or oncological indication. Examples of such inhibitors include, but are not limited to Erlotinib, Gefitinib, Lapatinib, Canertinib, BMS-599626 (AC-480), Neratinib, KRN-633, CEP-11981, Imatinib, Nilotinib, Dasatinib, AZM-475271, CP-724714, TAK-165, Sunitinib, Vatalanib, CP- 547632, Vandetanib, Bosutinib, Lestaurtinib, Tandutinib, Midostaurin, Enzastaurin, AEE-788, Pazopanib, Axitinib, Motasenib, OSI-930, Cediranib, KRN-951, Dovitinib, Seliciclib, SNS- 032, PD-0332991, MKC-I (Ro-3 l7453; R-440), Sorafenib, ABT-869, Brivanib (BMS- 582664), SU-14813, Telatinib, SU-6668, (TSU-68), L-21649, MLN-8054, AEW-541, and PD- 0325901.

EGFR tyrosine kinase inhibitors as used herein include, but are not limited to compounds selected from the group consisting of but not limited to Erlotinib, lapatinib, Rociletinib (CO- 1686), gefitinib, Dacomitinib (PF-00299804), Afatanib, Brigatinib (AP26113), WJTOG3405, NEJ002, AZD9291, HM61713, EGF816, ASP 8273, AC 0010. Examples of antibody EGFR inhibitors include Cetuximab, panitumumab, matuzumab, zalutumumab, nimotuzumab, necitumumab, Imgatuzumab (GA201, RO5083945), and ABT- 806.

The term "chemotherapeutic agent" has its general meaning in the art and refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolo melamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Intl. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-l l ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

The term“anti-cancer treatment” or“anti-cancer therapy” also refers to targeted cancer therapy. Targeted cancer therapies are drugs or other substances that block the growth and spread of cancer by interfering with specific molecules ("molecular targets") that are involved in the growth, progression, and spread of cancer. Targeted cancer therapies are sometimes called "molecularly targeted drugs", "molecularly targeted therapies", "precision medicines", or similar names. In some embodiments, the targeted therapy consists of administering the subject with a tyrosine kinase inhibitor as defined above.

The term “anti-cancer treatment” or “anti-cancer therapy” also refers to immunotherapeutic agent. The term "immunotherapeutic agent" as used herein, refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response against cancer cells and/or that decreases the side effects of other anticancer therapies. Immunotherapy is thus a therapy that directly or indirectly stimulates or enhances the immune system's responses to cancer cells and/or lessens the side effects that may have been caused by other anti-cancer agents. Immunotherapy is also referred to in the art as immunologic therapy, biological therapy biological response modifier therapy and biotherapy. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively, the immunotherapeutic treatment may consist of administering the subject with an amount of immune cells (T cells, NK, cells, dendritic cells, B cells...). Immunotherapeutic agents can be non-specific, i.e. boost the immune system generally so that the human body becomes more effective in fighting the growth and/or spread of cancer cells, or they can be specific, i.e. targeted to the cancer cells themselves immunotherapy regimens may combine the use of non-specific and specific immunotherapeutic agents. Non-specific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Non-specific immunotherapeutic agents have been used alone as a main therapy for the treatment of cancer, as well as in addition to a main therapy, in which case the non-specific immunotherapeutic agent functions as an adjuvant to enhance the effectiveness of other therapies (e.g. cancer vaccines). Non-specific immunotherapeutic agents can also function in this latter context to reduce the side effects of other therapies, for example, bone marrow suppression induced by certain chemotherapeutic agents. Non-specific immunotherapeutic agents can act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agents can themselves comprise cytokines. Non specific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. A number of cytokines have found application in the treatment of cancer either as general non-specific immunotherapies designed to boost the immune system, or as adjuvants provided with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins and colony- stimulating factors. Interferons (IFNs) contemplated by the present invention include the common types of IFNs, IFN-alpha (IFN-a), and IFN-beta (IFN-b). IFNs can act directly on cancer cells, for example, by slowing their growth, promoting their development into cells with more normal behaviour and/or increasing their production of antigens thus making the cancer cells easier for the immune system to recognise and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing down angiogenesis, boosting the immune system and/or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-alpha is available commercially as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). Interleukins contemplated by the present invention include IL-2, IL-4, IL-l l and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL- 12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present invention. Colony-stimulating factors (CSLs) contemplated by the present invention include sargramostim. Treatment with one or more growth factors can help to stimulate the generation of new blood cells in subjects undergoing traditional chemotherapy. Accordingly, treatment with CSLs can be helpful in decreasing the side effects associated with chemotherapy and can allow for higher doses of chemotherapeutic agents to be used. In addition to having specific or non-specific targets, immunotherapeutic agents can be active, i.e. stimulate the body's own immune response, or they can be passive, i.e. comprise immune system components that were generated external to the body. Passive specific immunotherapy typically involves the use of one or more monoclonal antibodies that are specific for a particular antigen found on the surface of a cancer cell or that are specific for a particular cell growth factor. Monoclonal antibodies may be used in the treatment of cancer in a number of ways, for example, to enhance a subject’s immune response to a specific type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors, such as those involved in angiogenesis, or by enhancing the delivery of other anticancer agents to cancer cells when linked or conjugated to agents such as chemotherapeutic agents, radioactive particles or toxins. Monoclonal antibodies currently used as cancer immunotherapeutic agents that are suitable for inclusion in the combinations of the present invention include, but are not limited to, rituximab (Rituxan®), trastuzumab (Herceptin®), ibritumomab tiuxetan (Zevalin®), tositumomab (Bexxar®), cetuximab (C-225, Erbitux®), bevacizumab (Avastin®), gemtuzumab ozogamicin (Mylotarg®), alemtuzumab (Campath®), and BL22. Other examples include anti-CTLA4 antibodies (e.g. Ipilimumab), anti-PDl antibodies, anti-PDLl antibodies, anti-TIMP3 antibodies, anti-LAG3 antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies or anti-B7H6 antibodies. In some embodiments, antibodies include B cell depleting antibodies. Typical B cell depleting antibodies include but are not limited to anti-CD20 monoclonal antibodies [e.g. Rituximab (Roche), Ibritumomab tiuxetan (Bayer Schering), Tositumomab (GlaxoSmithKline), AME-l33v (Applied Molecular Evolution), Ocrelizumab (Roche), Ofatumumab (HuMax-CD20, Gemnab), TRU-015 (Trubion) and IMMU-106 (Immuno medics)], an anti-CD22 antibody [e.g. Epratuzumab, Leonard et al, Clinical Cancer Research (Z004) 10: 53Z7-5334], anti-CD79a antibodies, anti-CD27 antibodies, or anti-CDl9 antibodies (e.g. U.S. Pat. No. 7,109,304), anti-BAFF-R antibodies (e.g. Belimumab, GlaxoSmithKline), anti- APRIL antibodies (e.g. anti-human APRIL antibody, ProSci inc.), and anti-IL-6 antibodies [e.g. previously described by De Benedetti et al, J Immunol (2001) 166: 4334-4340 and by Suzuki et al, Europ J of Immunol (1992) 22 (8) 1989-1993, fully incorporated herein by reference]. The immunotherapeutic treatment may consist of allografting, in particular, allograft with hematopoietic stem cell HSC. The immunotherapeutic treatment may also consist in an adoptive immunotherapy as described by Nicholas P. Restifo, Mark E. Dudley and Steven A. Rosenberg“Adoptive immunotherapy for cancer: harnessing the T cell response, Nature Reviews Immunology, Volume 12, April 2012). In adoptive immunotherapy, the subject’s circulating lymphocytes, NK cells, are isolated amplified in vitro and readministered to the subject. The activated lymphocytes or NK cells are most particularly be the subject’s own cells that were earlier isolated from a blood or tumor sample and activated (or“expanded”) in vitro.

The term“anti-cancer treatment” or“anti-cancer therapy” also refers to BRAF inhibitors such as vemurafenib, dacarbazine, dabrafenib, BMS-908662, LGX818, PLX3603, RAF265, R05185426, GSK2118436 and compounds described in Morris and Kopetz, 2013.

The term“anti-cancer treatment” or“anti-cancer therapy” also refers to radiotherapeutic agent. The term "radiotherapeutic agent" as used herein, is intended to refer to any radiotherapeutic agent known to one of skill in the art to be effective to treat or ameliorate cancer, without limitation. For instance, the radiotherapeutic agent can be an agent such as those administered in brachytherapy or radionuclide therapy such as Ra223 or Pb2l2. Such methods can optionally further comprise the administration of one or more additional cancer therapies, such as, but not limited to, chemotherapies, and/or another radiotherapy.

In one embodiment, said active compounds may be contained in the same composition or administrated separately. In a further aspect, the present invention relates to a compound selected from the group consisting of IL-10 inhibitor, TGFp i inhibitor and/or FOXP3 modulator for use in the treatment of progressive chronic lymphocytic leukemia in a patient in need thereof

In some embodiments, the present invention relates to a compound selected from the group consisting of IL-10 inhibitor, TGFp i inhibitor and/or FOXP3 modulator for use in the treatment of progressive chronic lymphocytic leukemia in a patient in need thereof wherein the patient was being classified as having or at risk of having or developing progressive chronic lymphocytic leukemia by the method as above described.

In some embodiments, the invention relates to combined preparation of the compounds of the invention for simultaneous, separate or sequential use in the treatment of progressive chronic lymphocytic leukemia in a patient in need thereof

Typically, 1, 2 or 3 compounds selected from the group consisting of IL-10 inhibitor, TGFp 1 inhibitor and FOXP3 modulator is used according to the invention.

In some embodiments, the present invention relates to an IL-10 inhibitor for use according to the invention.

In some embodiments, the present invention relates to a TGFp i inhibitor for use according to the invention.

In some embodiments, the present invention relates to a FOXP3 modulator for use according to the invention.

In some embodiments, the present invention relates to an IL-10 inhibitor and TGFp 1 inhibitor for use according to the invention.

In some embodiments, the present invention relates to an IL-10 inhibitor and FOXP3 modulator for use according to the invention.

In some embodiments, the present invention relates to a TGFp i inhibitor and FOXP3 modulator for use according to the invention.

In some embodiments, the present invention relates to an IL-10 inhibitor, TGFp 1 inhibitor and FOXP3 modulator for use according to the invention.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).

The term“modulator” has its general meaning in the art and refers to a target inhibitor or activator. The term“modulator” also refers to a compound that increase or decrease the expression of a specific gene.

The term“inhibitor” has its general meaning in the art and refers to a compound that selectively blocks or inactivates the target (IL-10, TGFp i and/or FOXP3). The term“inhibitor” also refers to a compound that selectively blocks the binding of the target to its substrate. The term“inhibitor” also refers to a compound able to prevent the action of the target for example by inhibiting the target controls of downstream effectors such as inhibiting the activation of the target pathway signalling. As used herein, the term“selectively blocks or inactivates” refers to a compound that preferentially binds to and blocks or inactivates the target with a greater affinity and potency, respectively, than its interaction with the other sub-types of the target family. Compounds that block or inactivate the target, but that may also block or inactivate other target sub-types, as partial or full inhibitors, are contemplated. The term“inhibitor” also refers to a compound that inhibits the target expression. Typically, an inhibitor is a small organic molecule, a polypeptide, an aptamer, an antibody, an oligonucleotide or a ribozyme. Tests and assays for determining whether a compound is a modulator, an inhibitor or an activator are well known by the skilled person in the art such as described in (Llorente et al, 2000; Clark et al, 2013; WO 2011/143280; US2013/0109619; WO2016196178; WO 2015/124715; US 2009/0325868).

The term“activator” has its general meaning in the art and refers to any compound that can directly or indirectly stimulate the signal transduction cascade related to the target (FOXP3). The term“activator” also refers to a compound that selectively activates the target. Typically, a FOXP3 activator is a small organic molecule, a peptide, a modified FOXP3 or an activator of FOXP3 expression.

The term“expression” when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs, which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP- ribosylation, myristilation, and glycosylation.

An“inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.

An“activator of expression” refers to a natural or synthetic compound that has a biological effect to activate the expression of a gene.

The term“IL-10 inhibitor” has its general meaning in the art and refers to compounds such as B-N10; ABF 13; B-S10; anti-IL-lO monoclonal antibody; IL-10 receptor antagonists; IL-10 receptor expression inhibitors and compounds described in Llorente et al, 2000; Clark et al, 2013; WO2011/143280; US2013/0109619; WO2016196178.

The term“TGFP inhibitor” has its general meaning in the art and refers to compounds such as Galunisertib (LY2157299); TEW-7197; LY3022859; IMC-TRI; Fresolimumab (GC- 1008); PF-03446962; Trabedersen (AP- 12009); Belagenpumatucel-L; Pirfenidone; and compounds described in Herbertz et al, 2015; W02010/089443; US2011/0294734; US2012/0315256; US2007/0014767; US2013/0225655.

The term “FOXP3 modulator” has its general meaning in the art and refers to compounds such as histone/protein deacetylases (HDAC), histone/protein deacetylases inhibitors (HDACi) and polypeptides and compounds described in US2010/0034786; WO2013/050596; Casares et al., 2010; Lozano et al., 2017. In another embodiment, the compound of the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then after raising aptamers directed against the target of the invention as above described, the skilled man in the art can easily select those blocking or inactivating the target.

In another embodiment, the compound of the invention is an antibody (the term including“antibody portion”) directed against the target and which is an IL-10 inhibitor, TGFp 1 inhibitor or a FOXP3 modulator.

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of the target. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes.

Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in the target. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation. Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modem Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab’)2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated a Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc’ regions to produce a functional antibody.

This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may be used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization.

In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et ah, /. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans.

In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non human sequences. The present invention also includes so-called single chain antibodies.

The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. In a preferred embodiment, the compound of the invention is a Human IgG4.

In another embodiment, the antibody according to the invention is a single domain antibody. The term“single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called“nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term“VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term“complementarity determining region” or“CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH. The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen- specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the“Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The“Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

In another aspect, the invention provides an antibody that competes for binding to the target with the antibody of the invention.

As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen or epitope typically is a binding with an affinity corresponding to a KD of about 10-7 M or less, such as about 10-8 M or less, such as about 10-9 M or less, about 10- 10 M or less, or about 10-11 M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats that are routinely used to epitope bin panels of monoclonal antibodies. Typically, an antibody binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten- fold lower, such as at least lOO-fold lower, for instance at least 1, 000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than its KD for binding to a non-specific antigen (e.g., BSA, casein), which is not identical or closely related to the predetermined antigen. When the KD of the antibody is very low (that is, the antibody has a high affinity), then the KD with which it binds the antigen is typically at least 10,000-fold lower than its KD for a non-specific antigen. An antibody is said to essentially not bind an antigen or epitope if such binding is either not detectable (using, for example, plasmon resonance (SPR) technology in a BIAcore 3000 instrument using a soluble form of the antigen as the ligand and the antibody as the analyte), or is 100 fold, 500 fold, 1000 fold or more than 1000 fold less than the binding detected by that antibody and an antigen or epitope having a different chemical structure or amino acid sequence.

Additional antibodies can be identified based on their ability to cross-compete (e.g., to competitively inhibit the binding of, in a statistically significant manner) with other antibodies of the invention in standard antigen binding assays. The ability of a test antibody to inhibit the binding of antibodies of the present invention to the target demonstrates that the test antibody can compete with that antibody for binding to the target; such an antibody may, according to non-limiting theory, bind to the same or a related (e.g., a structurally similar or spatially proximal) epitope on the target as the antibody with which it competes. Thus, another aspect of the invention provides antibodies that bind to the same antigen as, and compete with, the antibodies disclosed herein. As used herein, an antibody“competes” for binding when the competing antibody inhibits the target binding of an antibody or antigen binding fragment of the invention by more than

50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79, 80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98 or 99% in the presence of an equimolar concentration of competing antibody.

In other embodiments the antibodies or antigen binding fragments of the invention bind to one or more epitopes of the target. In some embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are linear epitopes. In other embodiments, the epitopes to which the present antibodies or antigen binding fragments bind are non-linear, conformational epitopes. In one embodiment, the compound of the invention is an IL-10 expression inhibitor, TGFp i expression inhibitor or a FOXP3 expression inhibitor.

The target expression inhibitors for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the target mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the target proteins, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding the target can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as target expression inhibitors for use in the present invention. The target gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that the target expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

Ribozymes can also function as target expression inhibitors for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of the target mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful target inhibitors can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3’ ends of the molecule, or the use of phosphorothioate or 2’-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing the target. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild- type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al, "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUCl8, pUCl9, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

Typically, the inhibitors according to the invention as described above are administered to the patient in a therapeutically effective amount.

By a "therapeutically effective amount" of the inhibitor of the present invention as above described is meant a sufficient amount of the inhibitor for treating cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the inhibitors and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific inhibitor employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific inhibitor employed; the duration of the treatment; drugs used in combination or coincidential with the specific inhibitor employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the inhibitor at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the inhibitor of the present invention for the symptomatic adjustment of the dosage to the patient to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the inhibitor of the present invention, particularly from 1 mg to about 100 mg of the inhibitor of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

In a particular embodiment, the compound according to the invention may be used in a concentration between 0.01 mM and 20 mM, particularly, the compound of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 mM. According to the invention, the compound of the present invention is administered to the subject in the form of a pharmaceutical composition. Typically, the compound of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The compound of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, the composition includes isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the compound of the present invention plus any additional desired ingredient from a previously sterile- filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In some embodiments, the compound of the present invention is administered sequentially or concomitantly with one or more therapeutic active agent such as anti-cancer therapy such as immunotherapeutic agent, chemotherapeutic agent or radiotherapeutic agent.

In one embodiment, said additional active compounds may be contained in the same composition or administrated separately.

In another embodiment, the pharmaceutical composition of the invention relates to combined preparation for simultaneous, separate or sequential use in the treatment of progressive chronic lymphocytic leukemia in a patient in need thereof.

The invention also provides kits comprising the compound of the invention. Kits containing the compound of the invention find use in therapeutic methods.

In a further aspect, the present invention relates to a method for treating progressive chronic lymphocytic leukemia in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound selected from the group consisting of IL-10 inhibitor, TGFp 1 inhibitor and/or FOXP3 modulator.

In a further aspect, the present invention relates to a method of treating progressive chronic lymphocytic leukemia in a patient in need thereof comprising the steps of:

(i) identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia by performing the method according to the invention, and

(ii) administering to said patient the compound of the invention and/or an anti-cancer treatment when it is concluded that the patient has or is at risk of having or developing progressive chronic lymphocytic leukemia.

In a further aspect, the present invention relates to a method of screening a candidate compound for use as a drug for treating cancer in a patient in need thereof, wherein the method comprises the steps of:

providing a IL-10, TGFp i and/or FOXP3, providing a cell, B cell, chronic lymphocytic leukemia B cells, bone marrow sample, lymphoid organ sample, blood sample, serum sample and plasma sample, tissue sample or organism expressing IL-10, TGFp i and/or FOXP3, providing a candidate compound such as a small organic molecule, a polypeptide, an aptamer, an antibody or an intra-antibody,

quantifying the IL-10, TGFp i and/or FOXP3 expression and/or activity, and selecting positively candidate compounds that inhibit IL-10, TGFp i and/or FOXP3 expression and/or activity, and/or that modulate FOXP3 expression and/or activity.

Methods for measuring IL-10, TGFp i and/or FOXP3 activity are well known in the art (Llorente et al, 2000; Clark et al, 2013; WO 2011/143280; US2013/0109619;

WO2016196178; WO 2015/124715 ; US 2009/0325868). For example, measuring the IL-10, TGFp 1 and/or FOXP3 activity involves determining a Ki on the IL-10, TGFp 1 and/or FOXP3 cloned and transfected in a stable manner into a CHO cell line, measuring cancer cells viability/survival, measuring cancer cell migration and invasion abilities, measuring cancer cell growth, measuring cancer cell proliferation, measuring cancer cell secretion in the presence or absence of the candidate compound.

Tests and assays for screening and determining whether a candidate compound is a IL- 10, TGFp i and/or FOXP3 inhibitor, and/or FOXP3 modulator are well known in the art (Llorente et al, 2000; Clark et al, 2013; WO 2011/143280; US2013/0109619;

WO2016196178; WO 2015/124715 ; US 2009/0325868; WO2010/089443; US2011/0294734; US2012/0315256; US2007/0014767; US2013/0225655; US2010/0034786; W02013/050596). In vitro and in vivo assays may be used to assess the potency and selectivity of the candidate compounds to inhibit IL-10, TGFp 1 and/or FOXP3 activity, or to modulate FOXP3 activity.

Activities of the candidate compounds, their ability to bind IL-10, TGFp 1 and/or FOXP3 and their ability to inhibit IL-10, TGFp 1 and/or FOXP3 activity, modulate FOXP3 activity may be tested using isolated cancer cell, cancer cell lines or CHO cell line cloned and transfected in a stable manner by the human IL-10, TGFp i and/or FOXP3.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: CLL-B cells express immune regulatory cytokines

A-C: Analysis of purified CD5⁺CDl9⁺ CLL-B cells from blood or lymph nodes (n=3) for IL10 and TGF 1 production by flow cytometry. (A and C) Representative dot plots with the percentage of positive populations, (B) Graphic representation of ILl0⁺ or TGF 1 cells among blood CD5⁺CDl9⁺ cells at the time of isolation (0, n=5) or after three days of culture (3, IL10 n=26, TGF 1 n=30) in the presence (+) or not (-) of stimulatory CD40L and anti-IgM. Dotted lines link individual patient samples. Wilcoxon signed-rank test. (C) Representative co expression of IL10 and TGF 1 . (D-E) Levels of the indicated cytokines secreted in the culture supernatant were evaluated by MSD technology (n=l7). (D right) Linear regression< between levels of TGF l in the culture supernatant and the percentage of TGF 1 cells is presented (r=0.82, n=9). Pearson’s correlation. (E) Cells were stimulated (+) or not (-) with CD40L and anti-IgM. Wilcoxon signed-rank test. *P< 0.05, ** P< 0.01; ns, not significant P >0.05.

Figure 2: CLL-B cells express FOXP3

(A-B): Analysis of purified CD5⁺CDl9⁺ CLL-B cells from blood or lymph nodes (n=3) for FOXP3 expression by flow cytometry. (A) Graphic representation of FOXP3⁺ cells among CD5⁺CDl9⁺ cells at the time of isolation (0, n=5) or after three days of culture (3) in the presence (+, n=34) or not (-, n=32) of CD40L and anti-IgM. Dotted lines link individual patient samples. (B). Representative co-expression of IL10 and FOXP3. The dotted line indicates that both sides are obtained from the same western blot. Wilcoxon signed-rank test, ns, not significant P >0.05.

Figure 3: Phenotypic Characterization of CLL-B cells.

(A-B) : Purified B cells were cultured for 72 hours and stained with the indicated membrane markers, and for IL-10, TGFp i or FOXP3. MFI of CD5, CD19, CD25, CD27 were compared between ILl0^+/ (n=l6), TGF-b I (n= 16) and FOXP3^+/ (n=l4) cells in unstimulated (A) and stimulated conditions (CD40L and anti-IgM, B). Ratio of membrane markers MFI between positive and negative cells is graphed. Wilcoxon signed-rank test * P<0.05, ** P<0.0l, ***P<0.00l,**** P<0.000l, ns, not significant P >0.05.

Figure 4: Differential expression of regulatory factors between CD5^h,ghCD19^h,gh and CD5^d,mCD19^d,m subpopulations

Purified B cells were stained for CD5 and CD 19 and analysed by flow cytometry. Cumulative results of IL10 (n=26), TGFp i (n=30) and FOXP3 (n=3 l) expression in CD5^highCDl9^high (H) and CD5^dimCDl9^dim (D) cells stimulated (+) or not (-), compared with Wilcoxon signed-rank test, * P<0.05, ** P<0.0l, ***P<0.00l,**** P<0.000l , ns, not significant P >0.05

Figure 5: Regulatory functions of CLL-B cells

(A) Purified CD4⁺T cells stimulated with anti-CD3 and anti-CD28 (S) or not (NS) were cultured for 48 hours in presence or not (-) with CLL-B cells activated with CD40L and anti- IgM at the indicated ratios (T/B). Scatter dot plots indicate the frequency of TNFa⁺ (left, n=5) and IFNy⁺ (right, n=9) cells among CD4⁺T cells. ANOVA test *p < 0.01; ** p<0.00l . (B) Bar graphs show the frequency of FOXP3⁺ among CD4⁺ T cells and CD4⁺CD25⁺CDl27^lowFOXP3⁺ Treg cells upon 48h co-culture (+) or not (-) with CLL-B cells (1 :1 ratio; n=5). Cells were stimulated (+) or not (-) as indicated. Wilcoxon signed-rank test * P<0.05, ** P<0.0l, ***P<0.00l . T cells stimulated (S) or not (NS) were co-cultured for 72h with CLL-B cells with the indicated ratios. Representative profile of 4 experiments is depicted.

Figure 6: A polyfunctional score is correlated with factors at risk of progression

Dimentional analysis of the polyfunctional score according to major risk factors: IGHV (Mutate, M; Unmutated, UM), ZAP70 and CD38 expressions (-, Negative and +, positive), Binet stage (A and B or C), and %MTS (Non responder, NR; Responder, R) of CLL progression in the CD5^hlghCDl9^hlgh subpopulation. Median and [Ql,Q3] are depicted. * P <0.05 (Mann- Whitney U test).

EXAMPLE:

Material & Methods

Patients, Materials and Methods

A cohort of 42 CLL patients was used in this study. CLL diagnosis was confirmed using international guidelines (43, 44) and approved by the local ethic committee (CLEA, GHPSSD, Avicenne hospital). The clinical and biological parameters (Service d’Hematologie Biologique de l’Hopital Avicenne) such as Binet stage at the experiment time, CD38 and ZAP-70 expressions, IGHV gene mutational status, cytogenetic features, TP53 mutation, other genetic mutations and need of treatment were analyzed (Tables 1-3).

Table 1. Patients’ biological and clinical parameters.

Table 2: CLL patients’s clinico-biological features

A. F: female and M: male

B. UM: UnMutated; M: Mutated

C. ND: not determined; POS: positive; NEG: negative

D. Metabolic activity in response to anti-IgM stimulation; R: Responder; NR: Non-Responder Table 3: CLL features and polyfunctional score

E. 0: no treatment at experiment time; 1 : treatment at the experiment

F. 0: A at the experiment time; 1 : B or C treatment at the experiment

G. 0: Zap70 negative; 1 : Zap70 positive

H. 0: IGHV mutated; 1 : IGHV unmutated

I. 0: CD38 negative; 1 : CD38 positive

J. 0: dell7 and/or dell l negative; 1 : dell7 and/or dell l positive

K. 0: TP53 WT; 1 : TP53 mutated

L. 0: no mutated; 1 : at least one mutated gene ( NOTCH1 , POT1, RPS15, SF3B1, BIRC3, FBW7, A TM, MYD88, PLCG2 ) Human cells isolation and cell culture

B and T CD4⁺ lymphocytes were isolated from total blood using Rosette B and CD4⁺ T isolation kits (STEMCELL). Isolated B and T cells subsets purity was assessed by flow cytometry analysis and was typically > 95%. Isolated B and T cells were cultured in RPMI 1640 containing L-glutamine and supplemented with 100 U/mg/ml penicillin/streptomycin (Life Technologies, USA), and 10% ECS (Biosera) at 37°C in humidified incubator containing 5% C02 at the concentration of 2xl0⁶/ml for 72 hours. B cells were stimulated or not with a combination of soluble CD40L (1 gg/ml; Miltenyi Biotec) and coated anti-IgM (20 gg/ml; Jackson ImmunoResearch) or a combination of soluble CD40L and IL21 (50ng/ml, Gibco by Life Technologies). CD4⁺ T cells were stimulated or not by coated anti-CD3 mAb (Hit-3a, 10 gg/ml; Ebioscience) and anti-CD28 mAb (CD28-2, 1 gg/ml; Ebio sciences). Lor cytokine detection by flow cytometry, Brefeldin A (BE A) (10 gg/ml; Sigma Aldrich), PMA (500 ng/ml; Sigma Aldrich) and Ionomycin (1 gg/ml; Sigma Aldrich) were added for the last 4 hours of culture. Lor B and CD4⁺ T cells co-culture experiments, B and T cells were mixed at different ratios (1 : 1, 1 :2, 1 :5 and 1 : 10) reaching a total cell number of 2x10⁶ cells. MTS was assessed as previously described (5, 6).

Cell sorting

Cell sorting was performed with purified CD5⁺CDl9⁺ peripheral blood CLL-B cells further labeled with PE-Vio770 anti-CD 19 (LT19), PerCP-Vio700 anti-CD3 (REA613), APC- Vio770 anti-CD5 (UCHT2) and VioBlue anti-CD27 (M-T271) (Miltenyi Biotec). After doublet cells exclusion, gating on CDl9⁺, CD3 , CD5⁺ and CD27^hlgh cells was performed. Sorted cells were then lysed to perform Western Blot. Cell-sorting experiments were performed on a LACS ARIA III cell sorter (BD Bioscience).

Western blotting

Sorted cells were lysed in NP40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1% NP-40, 10% Glycerol with protease inhibitors). Proteins (2 to 20 pg) were separated on a 10% SDS-PAGE, transferred on a nitrocellulose membrane and incubated with rabbit monoclonal anti-LOXP3 antibody (D608R, Cell Signaling Technologies) and with anti- b actin mAb (AC-74, Sigma Aldrich), followed by appropriate secondary horseradish peroxidase-conjugated antibody. Detection was performed using ECL kit (Bio-Rad) and images acquired with a Chemidoc MP (BioRad).

Flow cytometry

IL10, TϋRbI and LOXP3 CLL expressing cells were first labeled for extracellular staining with anti-CD 19/V500 (HIB19), anti-CD5/V450 (UCHT2), anti-CD24/FITC (ML5), anti-CD25/APC-Cy7 (M-A251), anti-CD27/PerCP5.5 (M-T271) and anti-CD38/PE-Cy7 (HIT2) mAbs (BD Biosciences) for 20 min. After washes, cells were fixed (2% PFA in PBS IX), permeabilized (0.5 % saponin, 1% BSA in PBS IX) and further stained with anti- IL10/APC (JES3-19F1), anti-FOXP3/PE (259D/C7) or anti-TGFp i/PE (TW4-9E7) mAbs or with their respective isotypes (BD Biosciences) overnight at 4°C.

In co-culture experiments the following alternative panel was used: anti-CD 19 A/500 (HIB19), CD5/FITC (UCHT2), CD25/APC-Cy7 (M-A251), CDl27/PE-Cy7 (SB/199) mAbs (BD Biosciences). Then, cells were fixed, permeabilized and stained with anti-IE 10/ APC (JES3-19F1), TGFp i/PE (TW4-9E7) or FOXP3/PE (259D/C7) (BD Biosciences).

For analysis of T cell division or TNFa and IFNy expression, CD4⁺ T cells were labeled with CellTrace Violet (Invitrogen) immediately after isolation according to the manufacter’s protocol. After co-culture for 72 hours, cells were labeled with anti-CDl9/PEVk>770 (LT19), CD3/PerCPVk>700 (REA613) and CD5/APCVio770 (UCHT2), mAbs (Miltenyi biotec) for 20 min. After washes, cells were fixed and permeabilized with cytofix/cytoperm buffer (BD biosciences) following the manufacturer’s protocol and then stained with anti-IFNy/PE (B27) (BD bioscience) and anti-TNFa/FITC (cA2) (Miltenyi biotech) or relevant isotype mAbs for 1 hour. Flow cytometry analysis was performed using a FACS Canto II driven by DIVA software (BD biosciences) and analyzed with the FlowJo software (Miltenyi Biotec).

Quantification of cytokine secretion in cell supernatants

Supernatants from B, CD4⁺ T and B/ CD4⁺ T cell cultures and co-cultures were frozen at -80°C and various cytokines (IF-l b, IF2, IF4, IF6, IF8, IF10, IF13, IF17, TNFa and IFNy, as well as TGFp 1 ) were quantified by V-plex assays (MSD) according to the manufacturer’s protocols.

Statistical analysis

Data are expressed as means with SEM or numbers with frequencies. For the comparison of qualitative values, the parametric chi-square test or Fisher's exact test was used and Mann- Whitney U test for the continuous variables or Wilcoxon signed-rank test (paired data). Association between cytokine secretion and producing cells was analyzed using Pearson correlation (r). Unsupervised hierarchical cluster analysis was performed using IF10, TGFp i and FOXP3 expression in unstimulated and stimulated state. An index called polyfunctional score was built by combining IF10, TGFp i and FOXP3 expression in each state (unstimulated or stimulated) using first component of principal components analysis. The assumption of unidimensionality of theses three factors was examined based on the eigenvalues plot (Data not shown). All tests were two-sided at a 0.05 significance level. Analyses were carried out using R statistical software version 3.1.2 and Graphpad (Prism 7).

Results

CLL-B cells express heterogeneous immunoregulatory cytokines profile.

Purified CLL-B cells, from a cohort of 42 CLL patients displaying variable biological parameters and clinical outcome (Tables 1-2) were analyzed for their ability to express two important immunosuppressive cytokines, IL10 and TGFp i . Flow cytometry analysis of CD5⁺CDl9⁺ cells at the time of isolation (DO) and after culture for 3 days (D3) showed a highly variable proportion of cells expressing IL10 or TGFp i ranging from 0.11 to 85% and from 0.22 to 71.1%, respectively (Figure 1A-B). Comparable frequencies of ILl0⁺ cells were found at the time of B cell isolation and in cultured cells indicative of an IL10 constitutive expression (22.95±4.83% at DO versus 27.54±5.76% at D3) (Figure 1B). By contrast, while very low levels of TGFpl expressing cells were observed at the time of isolation; several, but not all of the cases, increased TGFp 1 expressing cells in culture indicating a high degree of inducibility (2.86±l .89% at DO versus 25.66±4.92% at D3). In order to estimate the maximum extent of inducibility, cells were stimulated by addition of CD40L and anti-IgM during the 3 days of culture followed by Phorbol Myristate Acetate (PMA), ionomycin and brefelfinA for 4 h before analysis. Upon stimulation, for most of the samples, the proportion of IL10-expressing cells (27.54±5.76% versus 29.66±6.4%) was not modified, whereas further increase of TGFp i expressing cells (25.66±4.92% versus 33.39±6.24%) was observed (Figure 1A-B). Small needle puncture analysis confirmed the presence of such ILl0⁺ and TGFp i subpopulations in the lymph node (Figure 1A). Examination of individual cases analyzed in all conditions (connected lines) suggested both up or downregulation only in few cases for IL10. By contrast, several cases showed further induction ofTGFp i expressing cells upon stimulation while others remained at comparable levels (Figure 1B). Nonetheless, the intracellular levels of expression of the two cytokines revealed by the median of fluorescence of the cells was not modified upon stimulation (Data not shown) Indeed, flow cytometry on CD5⁺CDl9⁺ gated cells revealed a subset of cells co-expressing IL10 and TGFp 1 (Figure 1C).

Capacity to secrete various immune modulatory cytokines was also quantified. Cells in culture for 3 days and stimulated with CD40L/anti-IgM showed low levels of IL10 (mean = 0.5 pg/ml) and high levels of TGFp i (mean = 800 pg/ml). Interestingly, TGFp i was secreted at low or high levels allowing the segregation of two groups. Furthermore, the amount of secreted TGFp i in the high secreting group was correlated to the fraction of expressing cells with levels similar to healthy controls (Figure 1D and Data not shown). All the other cytokines tested were produced at low levels as compared to healthy controls. Similarly to TGF 1 , secretion of TNFa, IL8 and IL6 was induced upon stimulation while IL4, I L I b , IFNy , IL13, IL2 and IL17 were unchanged (Figure 1E and Data not shown). However, their production was not correlated to TGF 1 secretion (Data not shown). Granzyme B (GrB) expressing cells were rarely observed even when cells were triggered with a major inducer such as IL21/CD40L in contrast with B cells isolated from healthy subjects (Data not shown). Taken together, our results support the idea of a B-CLL cellular subpopulation, present at various extents among cases with a specific cytokine profile evocative of an immunoregulatory population that can further respond to BCR/CD40 stimulation.

CLL-B cells constitutively express FOXP3.

In line with the specific pattern of cytokines expressed, indicating similarities between regulatory and CLL-B cells, we assessed whether CLL-B cells could express, like Tregs, the major transcriptional regulator LOXP3. Llow cytometry analysis of peripheral viable CD5⁺CDl9⁺ CLL-B cells revealed a LOXP3⁺ subpopulation present at highly variable extents among patient samples in culture for 3 days (ranging from 0.04 to 82%; mean 20.76±3.79%) (Ligure 2A and Data not shown). Several LOXP3⁺ cells were also observed in small needle aspirate of lymph node and at collection of peripheral blood samples (mean 5.68±l . l9%). Stimulation for 3 days with CD40L/anti-IgM did not increase significantly the overall proportion of the LOXP3⁺ subpopulation (mean 21 56±4.36%) (Ligure 2A and Data not shown). Yet, a substantial up- or down-regulation of the LOXP3⁺ subpopulation was observed in several individual samples upon CD40L/anti-IgM triggering while others remained at comparable proportion (Ligure 2 A and Data not shown, connected lines). Mean fluorescence intensity of LOXP3 labeling did not change upon stimulation in CD5⁺CDl9⁺ patient B cells (Data not shown). Additionally, flow cytometry on CD5⁺CDl9⁺ gated cells revealed a subset of cells co expressing IL10 and LOXP3 (Ligure 2B). To confirm LOXP3 expression we performed a western blot analysis right after purification and further sorting or not using an antibody recognizing a different epitope of the transcription factor. Indeed, in CD3 CD5⁺CDl9⁺CD27⁺ sorted cells (Data not shown) the 47 kDa protein was expressed and enriched as compared to CD3 CD5⁺CDl9⁺ purified cells; LOXP3 was also observed in positive controls such as U20S or LOXP3 transfected HEK293T cells (Data not shown). Altogether, this analysis demonstrates that CLL-B cells display at variable extent a constitutive expression of LOXP3.

Specific subsets of CLL-B cells express IL-10, TGFpi and FOXP3 regulatory factors Next, we determined whether a particular CLL-B subtype could account for the expression of IL-10, TGFp i and FOXP3. Purified pathological B cells were split into IL-10, TGFp i and FOXP3 positive or negative subsets and were then assessed for the level of expression (MFI) of phenotypic markers (CD5, CD 19, CD27, CD24, CD25 and CD38), previously described in various regulatory subsets (Figure 3A-B and Data not shown). As shown in Figure 3 A, three phenotypic markers e.g. CD5, CD 19 and CD27 presented significant differences of expression between IL-l0⁺ and IL10 cells. Comparable results were obtained between TGFpl⁺ and TGFpl cells. Therefore, the memory CD5⁺CDl9⁺CD27⁺ CLL-B cells are the IL10 and/or TGFp 1 producing subsets. We also observed that FOXP3⁺ cells express to a certain extent higher levels of CD5, CD19 and CD25 compared to FOXP3 cells. Analysis upon CD40/BCR triggering confirmed the results obtained with unstimulated cells in spite of a weaker CD5 detection (Figure 3B). By contrast, several other phenotypic markers usually associated to IL10 producing cells were not differentially expressed between IL-l0^+/ , TGFp i or FOXP3^+/ CLL-B cells and were not influenced by in vitro stimulation (Data not shown).

CD5/CD19 expression levels discriminate intra-individual heterogeneity for regulatory-markers.

In line with the specific and heterogeneous individual signature of the two phenotypic markers CD 19 and CD5 for all three regulatory factors, we distinguished for a number of samples two subpopulations of B cells at steady state: CD5^hlghCDl9^hlgh and CD5^dimCDl9^dim (Data not shown). On a set of annotated patients, both subpopulations were evaluated over a period of time. Analysis showed clearly two intra- individual profiles. Several samples exhibited remarkably homogeneous CD5 and CD 19 expression with similar CD5^hlghCDl9^hlgh and CD5^dimCDl9^dim frequencies over a long period of time (1 to two years, Group 1, Data not shown). In contrast, a second group of samples showed a pronounced heterogeneity of CD5 and CD 19 expression and a significant variation of the CD5^hlghCDl9^hlgh and CD5^dimCDl9^dim frequencies even over a shorter period of time (less than a year, Group 2, Data not shown).

Importantly, the evaluation of ILlO, TGFp i and FOXP3 expressing cells showed striking differences for every regulatory factor between the two subpopulations, both in unstimulated or CD40/BCR stimulated cells (Figure 4). This analysis defined the CD5^hlghCDl9^hlgh subpopulation as the major source of the regulatory factors among CLL-B cells. In comparison the CD5^dimCDl9^dim subset exhibited a lower ability to express IL10, TGFp i and FOXP3 (Figure 4). Altogether, these results outline the heterogeneous profile of CLL cells within a single case and a particular subset, defined as CD5^hlghCDl9^hlgh B cells showing high expression levels of all three regulatory factors IL10, TGFp i and FOXP3 and diverging from already described regulatory B cells subsets.

CLL-B cells undergo a regulatory crosstalk with their T cell counterparts.

Next, we examined whether CLL-B cells might exhibit regulatory properties able to influence other immune cells. Purified autologous CD4⁺ T cells were cultured alone or together with CLL-B cells and their capacity to produce proinflammatory cytokines and regulatory factors was analyzed. The presence of stimulated CLL-B cells diminished, the percentage of CD4⁺ T cells expressing two important type 1 cytokines TNFa and IFNy. This effect was dependent on the T/ B cells ratio in the coculture (Figure 5A). Conversely, coculture between autologous T and CLL-B cells, increased the number of CD4⁺T cells expressing FOXP3. Since stimulation of T cells alone did not, this result argues for an orientation toward Tregs in the coculture (Figure 5B left). The modulation of FOXP3⁺ T cells was further supported by the assessment of CD4⁺CD25⁺CDl27^lowFOXP3⁺ Tregs, which was significantly enhanced in the presence of CLL-B cells (Figure 5B right).

Finally, co-culture of autologous CD4⁺ T cells with CLL-B cells clearly impacted T cells that have a low division rate in CLL. While up to 2 division cycles were observed for anti- CD3/CD28 stimulated T cells alone during 72 h, the presence of CLL-B cells, dependent on the ratio between B and T cells in the co-culture, markedly reduced T cells division rate with full blockade for a 1 :5 ratio (Data not shown). Altogether these results indicate a positive regulatory role of CLL-B cells toward the induction of CD4⁺ Tregs and at contrary a suppressive role on Thl cellular response.

Expression of IL-10, TGFpi and FOXP3 in CLL-B cells correlates with disease progression.

Following the characterization of these immune regulators, we performed an unsupervised hierarchical cluster analysis of the biomarkers among the various CD5⁺CDl9⁺ patient samples. First, using the frequency of IL10, TGFp i and FOXP3 expressing cells in unstimulated and stimulated conditions, we confirmed that stimulation influenced significantly the expression of TGFpl in a number of samples. Next, the hierarchical analysis of the three parameters in both conditions showed a strong correlation between the frequency ofTGFp 1 and FOXP3 expressing cells (r=0.8) and to a lesser extent with IL10 (r=0.6). Additionally, the analysis allowed the discrimination of two clusters of samples showing, respectively, weak or elevated occurrence for the three factors. Under stimulated conditions, the variables were distributed with a higher homogeneity individualizing a cluster positive for the three parameters (Data not shown). Taking separately into account the three factors, a statistical analysis did not reveal significant correlation with known risk factors of progression except for TGFp i that was elevated in samples positive for Zap70 (Data not shown). Next, we combined IL-10, TGFp 1 and FOXP3 expressions in a polyfunctional score, reflecting CLL-B cells capacity to express all 3 immunoregulatory factors. We challenged this score with the global or low and high CD5⁺CDl9⁺ subpopulations. The unidimensional validity of this polyfunctional score was confirmed with eigenvalues of components 2 and 3 lower than 1 in both unstimulated or stimulated conditions (Data not shown). Applying this polyfunctional score to the distinctive CD5^hlghCDl9^hlgh subpopulation (Figure 6) we found that values of polyfunctional score were significantly associated with IGHV status as well as with ZAP-70 positivity. These values were more elevated in IGHV unmutated or ZAP-70 positive cells. A similar analysis on CD5^lowCDl9^low subset or on global CLL population associated the score with Zap70 expression only.

By contrast, the novel regulatory score was never correlated with Binet staging of the disease at diagnosis, CD38 expression or metabolic activity (Figure 6 and Data not shown) (5, 6).

Remarkably, all the patients for whom the frequencies of the immonumodulating subpopulations were low (upper cluster of cases, Data not shown) experienced an indolent disease that did not require treatment. Their negative polyfunctional score was weakly associated with factors at risk of progression (20%, Table 3, UPN 14, 28, 20, 21, 24, 13). At the opposite, cases with high frequency of immuno modulating subpopulations (lower cluster of cases, Data not shown) presented with a progressive disease requiring treatment. Their positive polyfunctional score was majorly associated with factors at risk of progression (58%, Table 3, UPN 1, 23, 6, 22, 25). Moreover, a longitudinal analysis for several patients confirmed that the polyfunctional score was negative and stable for patients with an indolent disease (UPN 14, 28 and 13) while those experiencing a progression showed rise of the immunoregulatory score (UPN 2 and 8). Altogether the striking link between the capacities to detect higher levels of the three immunoregulatory factors in the tumor clone and the progressive profile of the patients argues for the validation of the polyfunctional score as a novel independent indicator of risk of progression. It also gives insights into the immunoregulatory capacity of the leukemic population.

Discussion

Recent advances in understanding the pathophysiology of CLL and the related heterogeneity at risk of progression have established the implication of dysregulated immune survey. The present study reveals important immune modulating functions of a subset of the clonal B-CLL expansion strongly correlated to disease progression. The analysis of a cohort of 42 patients has uncovered the capacity for CD5⁺CDl9⁺CD27⁺ memory CLL-B cells to produce and secrete not only constitutive IL10 but also inducible TGFp 1 . An important association between the level of expression of the phenotypic markers and the heterogeneous production of the cytokines defined two subpopulations; evolution of these subpopulations is evidenced for patients encountering disease progression. Interestingly, CD5^hlCDl9^hl CLL-B cells also produce at variable extent the transcription factor FOXP3, a hallmark of Tregs. Co-cultures with autologous T cells have proved the functional regulatory implication of CLL-B cells in the induction of Tregs, the negative regulation of CD4⁺ T cell proliferation, altogether with a modulation of their pattern of secretion toward deficiency of tumor clearance. Importantly, computation of the expression of the three regulatory factors in a polyfunctional score argues for its validation as a functional predictive marker of disease progression.

Several reports have suggested implication of IL10 producing CLL-B cells in the immune deficit in CD4⁺ T cells observed along disease progression (20, 21, 27). The major population characterized in the initial study had phenotypic features resembling those of CD 19⁺CD24^hlCD27⁺ B 10 cells, the human counterpart of murine CD 1 d^hlCD5⁺CD 19^hl B 10 cells (27). CD5⁺CDl9⁺CD24^hlCD27⁺ cells formed a relevant subset in the CLL-B cells of our cohort (Data not shown). However, the proportions of IL-l0⁺, TGFp i and FOXP3⁺ cells in this subset were not significantly different from those found in CD24^mterCD27⁺ or CD24⁺CD27 cells. In addition, both CD5⁺CD 19⁺CD24^hiCD27⁺ and CD5⁺CD 19⁺CD24^hiCD38^hi subsets were able to produce IL-10 and TGFp i cytokines (Data not shown). Several other subtypes such as immature CD 19⁺CD24^hiCD38^hi, CD 1 d⁺CD 19⁺CD38⁺IgM⁺CD 147⁺GrB⁺ or CDl lb⁺CDl9⁺ cells have also been described as IL10 competent cells (18, 19, 27, 32, 34). The frequency of CD1 lb⁺ and CD24^hlCD38^hl cells among CDl9⁺CD5⁺ CLL-B cells was less than 1% and could not account for the IL-10, TGFp i and FOXP3 expressions observed (Data not shown). Also GrB⁺ cells did not represent a significant proportion of CLL-B cells and GrB expression was only slightly induced upon IL21/CD40L triggering as compared to healthy controls (Data not shown). Finally, mechanisms of suppression via IL10 production have also been ascribed to mature B cells that do not express typical phenotypic markers of CLL-B cells such as plasmablasts or Brl cells (18). Our results indicate that the major ILl0⁺ and TGFp i producing cells are memory CD5⁺CDl9⁺CD27⁺ that differ from the other described regulatory subtypes with markers such as CD25, CD24 and CD38. Importantly, CLL-B cells also showed differential propensity to induce expression of TGFp i , an immunoregulatory factor for Tregs induction and CD8⁺ T cell anergy. These two immunomodulation have also been documented in CLL (11, 35, 36). Moreover, TGF 1 expression, when not already at the highest levels, was induced in a BCR/CD40 dependent manner, mimicking T/B cells interactions. We have previously shown that establishment of a threshold response to BCR triggering is mandatory for CLL-B cell heterogeneous survival (5). This result places the signal emanating from antigenic stimulation and crosstalk with the surrounding T cells at the center of the mechanisms responsible for a specific cytokine pattern with regulatory properties. In agreement with this central role of the BCR, we observed induction of several other cytokines involved in the remodeling of the immune response in a pathological environment such as IL6, IL8 or TNFa. The cytokines that rather promote specific helper T cells responses and pro -inflammatory signals, such as I L I b , IL2, IL4 or IFNy , were not induced and produced at very low levels as compared to healthy controls. In the pathological context, TGF 1 was induced at elevated levels while its secretion was not linked to the other cytokines. These results underline the inflection of the cytokinic balance in favor of the expansion of regulatory populations. Of note, similar ILlO⁺ and TGFp i subtypes were observed in lymph node aspirates from CLL patients suggesting similar modulatory mechanisms in secondary lymphoid organs.

Interestingly, CLL-B cells and mainly CD5^hlghCDl9^hlgh cells expressed also the transcription factor FOXP3 regardless of stimulation. Given that this factor is a major indicator of Tregs and of their immunomodulatory functions, our results demonstrated that FOXP3 is a novel indicator in CLL-B cells. A previous report described FOXP3 as present only in pre- apoptotic B cells in healthy controls (17). Therefore, we comforted our finding using different approaches including viability marker in flow cytometry and cell sorting to exclude any apoptotic cells or Tregs contamination that could account for FOXP3 expression. In CD5⁺CDl9⁺ samples we observed a strong match between the productions of the two immunoregulators FOXP3 and TGFp i . Phenotypically, FOXP3⁺ B cells also expressed CD25, another key marker of Tregs, which has been observed in ILlO⁺ Brl cells with antigen specific suppressive functions (37). Whether some coordinated regulation of FOXP3, TGFp 1 , CD5 and CD25 expressions might occur in CLL-B cell subtypes remains to be determined. A relationship between expression of CD5 and IL10 was already documented in CLL-B cells involving STAT3 and NFAT2 activation (38). Moreover, flow cytometry analysis revealed subpopulations expressing both IL10 and TGFp i or IL10 and FOXP3. Actually, FOXP3 and STAT3 that is constitutively phosphorylated in CLL are interacting partners for the expression of IL10 in a subset of regulatory T cells and a common regulation of the various proteins should be hypothesized (39). The regulatory function of such CLL-B cells was demonstrated in co-culture experiments with autologous T cells. First, we observed a strong orientation of T cells toward a regulatory profile with the expansion of CD4⁺CD25⁺CDl27^lowFOXP3⁺ Tregs. This orientation might result from the secretion by CLL-B cells of the two main suppressive cytokines IL10 and TGFp i (26). This expansion was accompanied by a reduction of two major CD4⁺ T cell populations, e.g. expressing TNF-a and IFNy targets of regulatory B cells, tumor clearance and cytotoxic response (18, 19, 40). This suppressive mechanism was dependent on the ratio in the co-culture between T and B cells and reflecting the cellular distortion observed in CLL patients. Finally, CLL-B cells hindered or suppress, in a T/B ratio dependent manner again, CD4⁺ T cells division, a major hallmark of Bregs. Remarkably, our analysis of the three suppressive factors expression identified the CD5^hlghCDl9^hlgh subpopulation as the functional subset expressing the highest levels of IL10, TGF 1 and FOXP3. This CD5^hlghCDl9^hlgh subset allowed distinction of two groups of cases with differential evolution during patients follow up. A reasonable interpretation of this finding is that high levels of regulatory factors provoke exhaustion of tumor survey mechanisms and tumor escape. At the opposite, the lower levels seen in CD5^dimCDl9^dim do not generate warning signals toward tumor survey. Among the three suppressive factors, TGF 1 only was statistically linked to ZAP70 expression or to BCR/CD40 triggering in IGHV unmutated patient samples arguing for the involvement of BCR signaling in the induction of TGFpU cells. In agreement with this result induction of TGF 1 Bregs was reported upon stimulation with CTLA-4 by T cells (30). Using T cell-independent CpG stimulation IL10 expressing cells were increased in IGHV mutated cases (21). Besides, our results indicate the presence of a constitutive pool of ILl0⁺ cells that was not induced upon B/T triggering.

In contrast with an individual analysis of suppressive factors, recent studies have interestingly reported a combinatorial evaluation of various cytokines or factors as better indicators of a functional subset. For instance, the balance between IL10 and TNFa expression in CDl9⁺CD24^hlghCD38^hlgh Bregs was further associated with renal allograft rejection than individual evaluation (41). Similarly, a polyfunctional index for T cells assembling expression of various cytokines was shown efficient during infectious disease response (42). Our analysis proved the benefit of using such a combinatorial score for its possible association with indicators at risk of progression of CLL patients. Computation of the three factors allowed better understanding of CLL-B cell activation and more importantly of the induction of its regulatory functions. When faced to the cohort of patient biological annotations, the polyfunctional score of the CD5^hlghCDl9^hlgh subset correlated with two major indicators of B cell activation and antigenic recognition, e.g. IGHV mutational status and ZAP70 expression. Similarly, higher polyfunctional score was associated with higher number of pejorative factors and patient disease progression necessitating treatment. With a measure of three different regulatory factors, the additional information given by the polyfunctional score might therefore become a better orientation indicator of disease progression and lack of tumor clearance.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

1. Fabbri G, Dalla-Favera R. The molecular pathogenesis of chronic lymphocytic leukaemia. Nat Rev Cancer. 2016; 16(3): 145-62.

2. Riches JC, Ramsay AG, Gribben JG. T-cell function in chronic lymphocytic leukaemia. Semin Cancer Biol. 2010;20(6):431-8.

3. Rosenwald A, Alizadeh AA, Widhopf G, Simon R, Davis RE, Yu X, et al. Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia. J Exp Med. 2001 ; 194(11): 1639-47.

4. Klein U, Tu Y, Stolovitzky GA, Mattioli M, Cattoretti G, Husson H, et al. Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med. 2001 ; 194(11): 1625-38.

5. Le Roy C, Deglesne PA, Chevallier N, Beitar T, Eclache V, Quettier M, et al. The degree of BCR and NFAT activation predicts clinical outcomes in chronic lymphocytic leukemia. Blood. 20l2;l20(2):356-65.

6. Deglesne PA, Chevallier N, Letestu R, Baran-Marszak F, Beitar T, Salanoubat C, et al. Survival response to B-cell receptor ligation is restricted to progressive chronic lymphocytic leukemia cells irrespective of Zap70 expression. Cancer Res. 2006;66(l4):7158- 66.

7. Muzio M, Apollonio B, Scielzo C, Frenquelli M, Vandoni I, Boussiotis V, et al. Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a molecular signature of anergy. Blood. 2008;l 12(1): 188-95.

8. Marklin M, Heitmann JS, Fuchs AR, Truckenmuller FM, Gutknecht M, Bugl S, et al. NFAT2 is a critical regulator of the anergic phenotype in chronic lymphocytic leukaemia. Nat Commun. 20l7;8(l):755. 9. Kipps TJ, Stevenson FK, Wu CJ, Croce CM, Packham G, Wierda WG, et al. Chronic lymphocytic leukaemia. Nat Rev Dis Primers. 20l7;3: l7008.

10. Herndon TM, Chen SS, Saba NS, Valdez J, Emson C, Gatmaitan M, et al. Direct in vivo evidence for increased proliferation of CLL cells in lymph nodes compared to bone marrow and peripheral blood. Leukemia. 2017;31 (6): 1340-7.

11. Taylor JG, Gribben JG. Microenvironment abnormalities and lymphomagenesis: Immunological aspects. Semin Cancer Biol. 2015;34:36-45.

12. Podhorecka M, Dmoszynska A, Rolinski J, Wasik E. T type l/type 2 subsets balance in B-cell chronic lymphocytic leukemia— the three-color flow cytometry analysis. Leuk Res. 2002;26(7):657-60.

13. Jitschin R, Braun M, Buttner M, Dettmer-Wilde K, Bricks J, Berger J, et al. CLL- cells induce IDOhi CDl4+HLA-DRlo myeloid-derived suppressor cells that inhibit T-cell responses and promote TRegs. Blood. 20l4;l24(5):750-60.

14. Inoue S, Mai A, Dyer MJ, Cohen GM. Inhibition of histone deacetylase class I but not class II is critical for the sensitization of leukemic cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Cancer Res. 2006;66(l3):6785-92.

15. Gorosito Serran M, Fiocca Vemengo F, Beccaria CG, Acosta Rodriguez EV, Montes CL, Gruppi A. The regulatory role of B cells in autoimmunity, infections and cancer: Perspectives beyond IL10 production. FEBS Lett. 20l5;589(22):3362-9.

16. Rosser EC, Mauri C. Regulatory B cells: origin, phenotype, and function. Immunity. 20l5;42(4):607-l2.

17. Noh J, Choi WS, Noh G, Lee JH. Presence of Foxp3 -expressing CDl9(+)CD5(+) B Cells in Human Peripheral Blood Mononuclear Cells: Human CDl9(+)CD5(+)Foxp3(+) Regulatory B Cell (Breg). Immune Netw. 20l0;l0(6):247-9.

18. Matsumoto M, Baba A, Yokota T, Nishikawa H, Ohkawa Y, Kayama H, et al. Interleukin- lO-producing plasmablasts exert regulatory function in autoimmune inflammation. Immunity. 20l4;4l(6): 1040-51.

19. Blair PA, Norena LY, Flores-Borja F, Rawlings DJ, Isenberg DA, Ehrenstein MR, et al. CDl9(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients. Immunity. 20l0;32(l): l29-40.

20. DiLillo DJ, Weinberg JB, Yoshizaki A, Horikawa M, Bryant JM, Iwata Y, et al. Chronic lymphocytic leukemia and regulatory B cells share IL-10 competence and immunosuppressive function. Leukemia. 20l3;27(l): 170-82. 21. Drennan S, D'Avola A, Gao Y, Weigel C, Chrysostomou E, Steele AJ, et al. IL- 10 production by CLL cells is enhanced in the anergic IGHV mutated subset and associates with reduced DNA methylation of the IL10 locus. Leukemia. 2017.

22. Baumgarth N. The double life of a B-l cell: self-reactivity selects for protective effector functions. Nat Rev Immunol. 201 l;l l(l):34-46.

23. Horikawa M, Minard-Colin V, Matsushita T, Tedder TF. Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice. J Clin Invest. 2011 ;121(11):4268-80.

24. Carter NA, Vasconcellos R, Rosser EC, Tulone C, Munoz-Suano A, Kamanaka M, et al. Mice lacking endogenous IL-lO-producing regulatory B cells develop exacerbated disease and present with an increased frequency of Thl/Thl7 but a decrease in regulatory T cells. J Immunol. 2011 ; 186(10):5569-79.

25. Flores-Borja F, Bosma A, Ng D, Reddy V, Ehrenstein MR, Isenberg DA, et al. CDl9+CD24hiCD38hi B cells maintain regulatory T cells while limiting TH1 and TH17 differentiation. Sci Transl Med. 20l3;5(l73): l73ra23.

26. Mauri C, Menon M. The expanding family of regulatory B cells. Int Immunol. 20l5;27(l0):479-86.

27. Iwata Y, Matsushita T, Horikawa M, Dilillo DJ, Yanaba K, Venturi GM, et al. Characterization of a rare IL-lO-competent B-cell subset in humans that parallels mouse regulatory B10 cells. Blood. 2011 ; 1 17 (2) : 530-41.

28. Shalapour S, Font-Burgada J, Di Caro G, Zhong Z, Sanchez-Lopez E, Dhar D, et al. Immunosuppressive plasma cells impede T-cell-dependent immunogenic chemotherapy. Nature. 20l5;52l(7550):94-8.

29. Hagn M, Blackwell SE, Beyer T, Ebel V, Fabricius D, Lindner S, et al. B-CLL cells acquire APC- and CTL-like phenotypic characteristics after stimulation with CpG ODN and IL-21. Int Immunol. 20l4;26(7):383-95.

30. Nouel A, Pochard P, Simon Q, Segalen I, Le Meur Y, Pers JO, et al. B-Cells induce regulatory T cells through TGF-b/IϋO production in A CTLA-4 dependent manner. J Autoimmun. 2015;59:53-60.

31. Hamblin TJ, Oscier DG. Chronic lymphocytic leukaemia: the nature of the leukaemic cell. Blood Rev. l997;l 1(3): 119-28.

32. Griffin DO, Holodick NE, Rothstein TL. Human Bl cells in umbilical cord and adult peripheral blood express the novel phenotype CD20+ CD27+ CD43+ CD70-. J Exp Med. 2011 ;208(l):67-80. 33. Chiorazzi N, Ferrarini M. B cell chronic lymphocytic leukemia: lessons learned from studies of the B cell antigen receptor. Annu Rev Immunol. 2003 ;21 : 841-94.

34. Lindner S, Dahlke K, Sontheimer K, Hagn M, Kaltenmeier C, Barth TF, et al. Interleukin 21 -induced granzyme B-expressing B cells infiltrate tumors and regulate T cells. Cancer Res. 20l3;73(8):2468-79.

35. Parekh VV, Prasad DV, Banerjee PP, Joshi BN, Kumar A, Mishra GC. B cells activated by lipopolysaccharide, but not by anti-Ig and anti-CD40 antibody, induce anergy in CD8+ T cells: role of TGF-beta 1. J Immunol. 2003; 170(12):5897-911.

36. Jak M, Mous R, Remmerswaal EB, Spijker R, Jaspers A, Yague A, et al. Enhanced formation and survival of CD4+ CD25hi Foxp3+ T-cells in chronic lymphocytic leukemia. Leuk Lymphoma. 2009;50(5):788-80l .

37. Vadasz Z, Peri R, Eiza N, Slobodin G, Balbir-Gurman A, Toubi E. The Expansion of CD25 high IL-10 high FoxP3 high B Regulatory Cells Is in Association with SLE Disease Activity. J Immunol Res. 20l5;20l5:254245.

38. Garaud S, Morva A, Lemoine S, Hillion S, Bordron A, Pers JO, et al. CD5 promotes IL-10 production in chronic lymphocytic leukemia B cells through STAT3 and NFAT2 activation. J Immunol. 2011 ; 186(8):4835-44.

39. Hossain DM, Panda AK, Manna A, Mohanty S, Bhattachaqee P, Bhattacharyya S, et al. FoxP3 acts as a cotranscription factor with STAT3 in tumor-induced regulatory T cells. Immunity. 20l3;39(6): 1057-69.

40. Lemoine S, Morva A, Youinou P, Jamin C. Human T cells induce their own regulation through activation of B cells. J Autoimmun. 2011;36(3-4):228-38.

41. Cherukuri A, Rothstein DM, Clark B, Carter CR, Davison A, Hemandez- Fuentes M, et al. Immunologic human renal allograft injury associates with an altered IL- lO/TNF-a expression ratio in regulatory B cells. J Am Soc Nephrol. 20l4;25(7): 1575-85.

42. Larsen M, Arnaud L, Hie M, Parizot C, Dorgham K, Shoukry M, et al. Multiparameter grouping delineates heterogeneous populations of human IL-17 and/or IL-22 T-cell producers that share antigen specificities with other T-cell subsets. Eur J Immunol. 201 l;4l(9):2596-605.

43. Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;l 1 l(l2):5446-56. 44. Eichhorst B, Robak T, Montserrat E, Ghia P, Hillmen P, Hallek M, et al. Chronic lymphocytic leukaemia: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 20l5;26 Suppl 5:v78-84.

45. Llorente, et al., Clinical and biologic effects of antiinterleukin- 10 monoclonal antibody administration in systemic lupus erythematosus, Arthritis Rheum. 43 (8) (2000) 1790-

1800.

46. Clark DN, Markham JL, Sloan CS, Poole BD. Cytokine inhibition as a strategy for treating systemic lupus erythematosus. Clin Immunol. 2013 Sep;l48(3):335-43.

47. Casares N, Rudilla F, Arribillaga L, Llopiz D, Riezu-Boj JI, Lozano T, Lopez- Sagaseta J, Guembe L, Sarobe P, Prieto J, Borras-Cuesta F, Lasarte JJ. A peptide inhibitor of

FOXP3 impairs regulatory T cell activity and improves vaccine efficacy in mice. J Immunol. 2010 Nov 1 ; 185(9):5150-9.

48. Teresa Lozano, Marta Gorraiz, Aritz Lasarte-Cia, Marta Ruiz, Obdulia Rabal, Julen Oyarzabal, Sandra Hervas-Stubbs, Diana Llopiz, Pablo Sarobe, Jesus Prieto, Noelia Casares, and Juan Jose Lasarte. Blockage of FOXP3 transcription factor dimerization and FOXP3/AML1 interaction inhibits T regulatory cell activity: sequence optimization of a peptide inhibitor. Oncotarget. 2017 Sep 22; 8(42): 71709-71724.

Claims

CLAIMS:

1. A method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of:

i) determining the level of IL-10, TGFp i and FOXP3 or the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 in a biological sample obtained from the patient,

ii) comparing the levels determined at step i) with a reference value, and

iii) wherein detecting differential in the level of IL-10, TGFp i and FOXP3 or the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 between the biological sample and the reference value is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia.

2. A method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of:

i) determining the level of IL-10, TGFp i and FOXP3 or the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 in a biological sample obtained from the patient at time tl,

ii) determining the level of IL-10, TGFp i and FOXP3 or the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 in a biological sample obtained from the patient at time t2, and

iii) an increase of the levels determined at step i) and ii) between tl and t2 is indicative of patient having or at risk of having or developing progressive chronic lymphocytic leukemia or resisting to treatment.

3. A method of identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia comprising the steps of:

ii) determining the level of IL-10, TGFp i and FOXP3 or the level of cells expressing IL-10, cells expressing TGFp 1 , and cells expressing FOXP3 in a biological sample obtained from the patient at time t2, iii) building a polyfunctional score combining the levels determined at step i) and a polyfunctional score combining the levels determined at step ii), and

4. A compound selected from the group consisting of IL-10 inhibitor, TGFp i inhibitor and/or FOXP3 modulator for use in the treatment of progressive chronic lymphocytic leukemia in a patient in need thereof.

5. A compound selected from the group consisting of IL-10 inhibitor, TGFp i inhibitor and/or FOXP3 modulator for use in the treatment of progressive chronic lymphocytic leukemia in a patient in need thereof wherein the patient was being classified as having or at risk of having or developing progressive chronic lymphocytic leukemia by the method according to any of claims 1 to 3.

6. A method for treating progressive chronic lymphocytic leukemia in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound selected from the group consisting of IL-10 inhibitor, TGFp i inhibitor and/or FOXP3 modulator.

7. A method of treating progressive chronic lymphocytic leukemia in a patient in need thereof comprising the steps of:

(i) identifying a patient having or at risk of having or developing progressive chronic lymphocytic leukemia by performing the method according to any of claims 1 to 3, and

(ii) administering to said patient the compound according to claim 4 and/or an anti-cancer treatment when it is concluded that the patient has or is at risk of having or developing progressive chronic lymphocytic leukemia.